Substrate preparation process

ABSTRACT

The present invention provides novel processes for the large scale preparation of arrays of polymer sequences wherein each array includes a plurality of different, positionally distinct polymer sequences having known monomer sequences. The methods of the invention combine high throughput process steps with high resolution photolithographic techniques in the manufacture of polymer arrays.

This application is a continuation of U.S. application Ser. No.10/722,032, filed on Nov. 25, 2003, which is a continuation of U.S.application Ser. No. 09/716,507, filed Nov. 20, 2000; which is acontinuation of U.S. patent application Ser. No. 09/244,568. filed Feb.4, 1999, now U.S. Pat. No. 6,307,042; which in turn is a continuation ofU.S. patent application Ser. No. 08/634,053, filed Apr. 17, 1996, nowU.S. Pat. No. 5,959,098; all of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

Methods for synthesizing a variety of different types of polymers arewell known in the art. For example, the “Merrifield” method, describedin Atherton et al., “Solid Phase Peptide Synthesis,” IRL Press, 1989,which is incorporated herein by reference for all purposes, has beenused to synthesize peptides on a solid support. In the Merrifieldmethod, an amino acid is covalently bonded to a support made of aninsoluble polymer or other material. Another amino acid with an alphaprotecting group is reacted with the covalently bonded amino acid toform a dipeptide. After washing, the protecting group is removed and athird amino acid with an alpha protecting group is added to thedipeptide. This process is continued until a peptide of a desired lengthand sequence is obtained.

Methods have also been developed for producing large arrays of polymersequences on solid substrates. These large “arrays” of polymer sequenceshave wide ranging applications and are of substantial importance to thepharmaceutical, biotechnology and medical industries. For example, thearrays may be used in screening large numbers of molecules forbiological activity, i.e., receptor binding capability. Alternatively,arrays of oligonucleotide probes can be used to identify mutations inknown sequences, as well as in methods for de novo sequencing of targetnucleic acids.

Of particular note, is the pioneering work described in U.S. Pat. No.5,143,854 (Pirrung et al.) and PCT Application No. 92/10092 discloseimproved methods of molecular synthesis using light directed techniques.According to these methods, light is directed to selected regions of asubstrate to remove protecting groups from the selected regions of thesubstrate. Thereafter, selected molecules are coupled to the substrate,followed by additional irradiation and coupling steps. By activatingselected regions of the substrate and coupling selected monomers inprecise order, one can synthesize an array of molecules having anynumber of different sequences, where each different sequence is in adistinct, known location on the surface of the substrate.

These arrays clearly embody the next step in solid phase synthesis ofpolymeric molecules generally, and polypeptides and oligonucleotides,specifically. Accordingly, it would be desirable to provide methods forpreparation of these arrays, which methods have high throughput, highproduct quality, enhanced miniaturization and lower costs. The presentinvention meets these and other needs.

SUMMARY OF THE INVENTION

The present invention generally provides novel processes for theefficient, large scale preparation of arrays of polymer sequenceswherein each array includes a plurality of different, positionallydistinct polymer sequences having known monomer sequences. In oneembodiment, the methods of the present invention provide for thecleaning and stripping of substrate wafers to remove oil and dirt fromthe surface, followed by the derivatization of the wafers to providephotoprotected functional groups on the surface. Polymer sequences arethen synthesized on the surface of the substrate wafers by selectivelyexposing a plurality of selected regions on the surface to an activationradiation to remove the photolabile protecting groups from thefunctional groups and contacting the surface with a monomer containingsolution to couple monomers to the surface in the selected regions. Theexposure and contacting steps are repeated until a plurality of polymerarrays are formed on the surface of the substrate wafer. Each polymerarray includes a plurality of different polymer sequences coupled to thesurface of the substrate wafer in a different known location. The wafersare then separated into a plurality of individual substrate segments,each segment having at least one polymer array formed thereon, andpackaged in a cartridge whereby the surface of said substrate segmenthaving the polymer array formed thereon is in fluid contact with thecavity.

In another embodiment, the present invention provides methods of formingpolymer arrays by providing a substrate having a first surface coatedwith functional groups protected with a photolabile protecting group,and a second surface having a layer that includes one or more of anindex matching compound, a light absorbing compound and anantireflective compound. The method then provides for the sequentialactivation and coupling of monomers in different selected regions of thefirst surface of the substrate to form a plurality of different polymersequences in different known locations on the surface of the substrate,by directing an activation radiation at the first surface of thesubstrate.

In yet another embodiment, the present invention provides a method offorming a plurality of polymer arrays using a batch process. Inparticular, this method comprises the steps of activating a plurality ofsubstrate wafers by exposing selected regions on each of a plurality ofsubstrate wafers then contacting them with a monomer containing solutionin a batch.

In a further embodiment, the present invention provides a method ofsynthesizing polymers on substrates by first derivatizing the substratewith an aminoalkyltrialkoxysilane.

In an additional embodiment, the present invention provides a method forforming an array of polymers on a substrate using light-directedsynthesis wherein the exposing step comprises directing an activationradiation at selected regions on the surface of said substrate byshining the activation radiation through a photolithographic mask havingtransparent regions and opaque regions where the transparent regions aresmaller than the selected regions. As a result, the activation radiationshone through the transparent regions in the mask is diffracted toexpose the selected regions.

The present invention also provides methods of forming arrays of polymersequences having enhanced synthesis efficiencies through theincorporation of monomers which have lipophilic chemical groups coupledthereto.

The present invention also provides methods of forming polymer arraysusing the above-described methods, but wherein the deprotection andcoupling steps in adjacent selected regions of the substrate surface arealigned to minimize differences in synthesis steps between adjacentregions.

In still another embodiment, the present invention provides polymerarrays and methods of forming them on a tubular substrate by thesequential activation of and coupling of monomers to selected segmentsof the tubular substrate surface.

In an additional embodiment, the present invention provides methods ofphotoprotecting functional groups that are coupled to solid supports byexposing the functional group to a photoprotecting group transfer agenthaving the formula:

wherein R₁ is a photolabile protecting group and X is a leaving group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates light directed oligonucleotidesynthesis using photolithographic methods.

FIGS. 2A-C are flow diagrams illustrating the overall process ofsubstrate preparation. FIG. 2A is a flow dagram illustrating the overallprocess. FIGS. 2B and 2C are flow diagrams of the synthesis steps forindividual and batch processes, respectively.

FIGS. 3A and 3B show schematic illustrations of alternate reactorsystems for carrying out the combined photolysis/chemistry steps of usedin the methods of the present invention.

FIGS. 4A and 4B schematically illustrate different isolated views of aflow cell incorporated into the reactor systems of FIGS. 3A and 3B. FIG.4C shows a schematic illustration of an integrated reactor systemincluding computer control and substrate translation elements.

FIG. 5A shows the alkylation of the exocyclic amine functional group ofdeoxyguanosine with dimethoxytritylchloride (DMT-CI) and subsequentcoupling of a MenPOC protecting group to the 3′ hydroxyl group of anucleoside phosphoramidite. FIG. 5B shows the synthetic route forproduction of Fmoc-phosphoramidites. FIG. 5C shows a synthetic route forintroduction of a lipophilic substituent to the photoprotecting groupMeNPOC.

FIG. 6A shows a schematic representation of a device including a sixvessel reaction chamber bank, for carrying out multiple parallel monomeraddition steps separate from the photolysis step in light directedsynthesis of oligonucleotide arrays. FIG. 6B shows a detailed view of asingle reaction chamber.

FIG. 7 illustrates a substrate wafer fabricated with a plurality ofprobe arrays which wafer also includes alignment marks.

FIG. 8 illustrates one embodiment of an array cartridge into which anarray substrate is placed for use.

FIGS. 9A and 9B show the coupling of fluorescent nucleotides to asubstrate surface using photolithographic methods in 50 and 100 μmfeatures, using back-side and frontside exposure, respectively. FIGS. 9Cand 9D show a plot of fluorescence intensity as a function of substrateposition at the border between two features for back-side and front-sideexposure as indicated. FIG. 9C illustrates the contrast difference froma top view of the plots while FIG. 9D shows a side view.

FIG. 10 is a bar chart showing a comparison of silanation methods using5 different silanes to derivatize the surface of glass substrates(3-acetoxypropyltrimethoxysilane (“OAc”);3-glycidoxypropyltrimethoxysilane (“Epoxy”);4-(hydroxybutyramido)propyltriethoxysilane (“Mono”);3-aminopropyltriethoxysilane (“APS”); and3-N,N-bis(2-hydroxyethyl)aminopropyl; triethoxysilane (“bis”)). Shownare the surface density of reactive groups as shown by fluorescencestaining (black) and fluorescence intensity of a standard hybridizationexperiment following synthesis of oligonucleotides on the surface ofsubstrates derivatized using these silanes (grey).

FIG. 11 shows the reprotection of deprotected hydroxyl groups on a glasssubstrate with MeNPOC-tetrazolide as a function of time of exposure tothe MeNPOC-tetrazolide and addition of catalyst.

DESCRIPTION OF THE PREFERRED EMBODIMENT

I. Definitions

Probe: A probe, as defined herein, is a surface-immobilized moleculethat is recognized by a particular target. These may also be referred toas ligands. Examples of probes encompassed by the scope of thisinvention include, but are not limited to, agonists and antagonists ofcell surface receptors, toxins and venoms, viral epitopes, hormonereceptors, peptides, peptidomimetics, enzymes, enzyme substrates,cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins or monoclonal antibodies, natural ormodified, e.g., reshaped, chimeric, etc.

Array: An array is a preselected collection of different polymersequences or probes which are associated with a surface of a substrate.An array may include polymers of a given length having all possiblemonomer sequences made up of a specific basis set of monomers, or aspecific subset of such an array. For example, an array of all possibleoligonucleotides of length 8 includes 65,536 different sequences.However, as noted above, an oligonucleotide array also may include onlya subset of the complete set of probes. Similarly, a given array mayexist on more than one separate substrate, e.g., where the number ofsequences necessitates a larger surface area in order to include all ofthe desired polymer sequences.

Functional group: A functional group is a reactive chemical moietypresent on a given monomer, polymer or substrate surface. Examples offunctional groups include, e.g., the 3′ and 5′ hydroxyl groups ofnucleotides and nucleosides, as well as the reactive groups on thenucleobases of the nucleic acid monomers, e.g., the exocyclic aminegroup of guanosine, as well as amino and carboxyl groups on amino acidmonomers.

Monomer/Building block: A monomer or building block is a member of theset of smaller molecules which can be joined together to form a largermolecule or polymer. The set of monomers includes but is not restrictedto, for example, the set of common L-amino acids, the set of D-aminoacids, the set of natural or synthetic amino acids, the set ofnucleotides (both ribonucleotides and deoxyribonucleotides, natural andunnatural) and the set of pentoses and hexoses. As used herein, monomerrefers to any member of a basis set for synthesis of a larger molecule.A selected set of monomers forms a basis set of monomers. For example,the basis set of nucleotides includes A, T (or U), G and C. In anotherexample, dimers of the 20 naturally occurring L-amino acids form a basisset of 400 monomers for synthesis of polypeptides. Different basis setsof monomers may be used in any of the successive steps in the synthesisof a polymer. Furthermore, each of the sets may include protectedmembers which are modified after synthesis.

Feature: A feature is defined as a selected region on a surface of asubstrate in which a given polymer sequence is contained. Thus, where anarray contains, e.g., 100,000 different positionally distinct polymersequences on a single substrate, there will be 100,000 features.

Edge: An edge is defined as a boundary between two features on a surfaceof a substrate. The sharpness of this edge, in terms of reduced bleedover from one feature to another, is termed the “contrast” between thetwo features.

Protecting group: A protecting group is a material which is chemicallybound to a reactive functional group on a monomer unit or polymer andwhich protective group may be removed upon selective exposure to anactivator such as a chemical activator, or another activator, such aselectromagnetic radiation or light, especially ultraviolet and visiblelight. Protecting groups that are removable upon exposure toelectromagnetic radiation, and in particular light, are termed“photolabile protecting groups.”

II. ABBREVIATIONS

-   -   ACN Acetonitrile    -   Bz Benzoyl    -   CE β-cyanoethyl    -   CEP Cyanoethylphosphoramidite    -   DCM dichloromethane    -   DIEA Diiminoethylamine    -   dG Deoxyguanosine    -   DMAP Dimethylaminopyridine    -   DMC N,N-dimethylcarbamoyl    -   DMF Dimethylformamide    -   DMT 4,4′-Dimethoxytrityl    -   DPC Diphenylcarbamoyl    -   HOAT 1-Hydroxy-7-azabenzotriazole    -   HOBT 1-hydroxybenzotriazole    -   Ibu Isobutyryl    -   MeNP α-methyl-o-nitropiperonyl    -   MeNPOC α-methyl-o-nitropiperonyloxycarbonyl    -   MeNV α-methyl-o-nitroveratryl    -   MeNVOC α-methyl-o-nitroveratryloxycarbonyl    -   MMT 4-Methoxytrityl    -   NMI n-methylimidazole    -   NMP n-methylpyrollidinone    -   NP o-Nitropiperonyl    -   NPE 2-(p-nitrophenyl)ethyl    -   NPSE 2-(p-nitrophenylsulfonyl)ethyl    -   NV o-Nitroveratryl    -   NPOC o-Nitropiperanyloxycarbonyl    -   NVOC o-Nitroveratryloxycarbonyl    -   PAC Phenoxyacetyl    -   PYMOC 1-Pyrenylmethyloxycarbonyl    -   SSPE Saline, Sodium Phosphate, EDTA Buffer    -   TEA Triethylamine    -   THF Tetrahydrofuran        III. Process Overview

The present invention generally provides processes and devices forreproducibly and efficiently preparing arrays of polymer sequences onsolid substrates. The overall process is illustrated in FIG. 2A.Generally, the process 1 begins with a series of substrate preparationsteps 10 which may include such individual processing steps as strippingcleaning and derivatization of the substrate surface to provide uniformreactive surfaces for synthesis. The polymer sequences are thensynthesized on the substrate surface in the synthesis step 20. Followingpolymer synthesis, the substrates are then separated into individualarrays 40, and assembled in cartridges that are suitable for ultimateuse 60. In alternate embodiments, the present invention also providesfor the synthesis of the polymer sequences on the substrate surfaceusing either an individual or batch process mode. A comparison of thesetwo synthesis modes is shown in FIG. 2B. In the individual processingmode, the activation and monomer addition steps are combined in a singleunit operation 22. For example, a single substrate wafer is placed in areactor system where it is first subjected to an activation step toactivate selected regions of the substrate. The substrate is thencontacted with a first monomer which is coupled to the activated region.Activation and coupling steps are repeated until the desired array ofpolymer sequences is created. The arrays of polymer sequences are thensubjected to a final deprotection step 30.

In the batch processing mode, a number of substrate wafers are subjectedto an activating step 24. The activated substrate wafers are then pooled26 and subjected to a monomer addition step 28. Each substrate wafer isthen subjected individually to additional activation steps followed bypooling and monomer addition. This is repeated until a desired array ofpolymer sequences is formed on the substrate wafers in a series ofindividual arrays. These arrays of polymer sequences on the substratewafers are then subjected to a final deprotection step 30.

IV. Substrate Preparation

The term “substrate” refers to a material having a rigid or semi-rigidsurface. In many embodiments, at least one surface of the substrate willbe substantially flat or planar, although in some embodiments it may bedesirable to physically separate synthesis regions for differentpolymers with, for example, wells, raised regions, etched trenches, orthe like. According to other embodiments, small beads may be provided onthe surface which may be released upon completion of the synthesis.Preferred substrates generally comprise planar crystalline substratessuch as silica based substrates (e.g. glass, quartz, or the like), orcrystalline substrates used in, e.g., the semiconductor andmicroprocessor industries, such as silicon, gallium arsenide and thelike. These substrates are generally resistant to the variety ofsynthesis and analysis conditions to which they may be subjected.Particularly preferred substrates will be transparent to allow thephotolithographic exposure of the substrate from either direction.

Silica aerogels may also be used as substrates. Aerogel substrates maybe used as free standing substrates or as a surface coating for anotherrigid substrate support. Aerogel substrates provide the advantage oflarge surface area for polymer synthesis, e.g., 400 to 1000 m²/gm, or atotal useful surface area of 100 to 1000 cm² for a 1 cm² piece ofaerogel substrate. Such aerogel substrates may generally be prepared bymethods known in the art, e.g., the base catalyzed polymerization of(MeO)₄Si or (EtO)₄Si in ethanol/water solution at room temperature.Porosity may be adjusted by altering reaction coondition by methodsknown in the art.

Individual planar substrates generally exist as wafers which can havevaried dimensions. The term “wafer” generally refers to a substantiallyflat sample of substrate from which a plurality of individual arrays orchips may be fabricated. The term “array” or “chip” is used to refer tothe final product of the individual array of polymer sequences, having aplurality of different positionally distinct polymer sequences coupledto the surface of the substrate. The size of a substrate wafer isgenerally defined by the number and nature of arrays that will beproduced from the wafer. For example, more complex arrays, e.g., arrayshaving all possible polymer sequences produced from a basis set ofmonomers and having a given length, will generally utilize larger areasand thus employ larger substrates, whereas simpler arrays may employsmaller surface areas, and thus, less substrate.

Typically, the substrate wafer will range in size of from about 1″×1″ toabout 12″×12″, and will have a thickness of from about 0.5 mm to about 5mm. Individual substrate segments which include the individual arrays,or in some cases a desired collection of arrays, are typically muchsmaller than the wafers, measuring from about 0.2 cm×0.2 cm 20 to about5 cm×5 cm. In particularly preferred aspects, the substrate wafer isabout 5″×5″ whereas the substrate segment is approximately 1.28 cm×1.28cm. Although a wafer can be used to fabricate a single large substratesegment, typically, a large number of substrate segments will beprepared from a single wafer. For example, a wafer that is 5″×5″ can beused to fabricate upwards of 49 separate 1.28 cm×1.28 cm substratesegments. The number of segments prepared from a single wafer willgenerally vary depending upon the complexity of the array, and thedesired feature size.

Although primarily described in terms of flat or planar substrates, thepresent invention may also be practiced with substrates havingsubstantially different conformations. For example, the substrate mayexist as particles, strands, precipitates, gels, sheets, tubing,spheres, containers, capillaries, pads, slices, films, plates, slides,etc. In a preferred alternate embodiment, the substrate is a glass tubeor microcapillary. The capillary substrate provides advantages of highersurface area to volume ratios, reducing the amount of reagents necessaryfor synthesis. Similarly, the higher surface to volume ratio of thesecapillary substrates imparts more efficient thermal transfer properties.Additionally, preparation of the polymer arrays may be simplifiedthrough the use of these capillary based substrates. For example,minimizing differences between the regions on the array, or “cells”, andtheir “neighboring cells” is simplified in that there are only twoneighboring cells for any given cell (see discussion below for edgeminimization in chip design). Spatial separation of two neighboringcells on an array merely involves the incorporation of a single blankcell, as opposed to full blank lanes as generally used in a flatsubstrate conformation. This substantially conserves the surface areaavailable for polymer synthesis. Manufacturing design may also besimplified by the linear nature of the substrate. In particular, thelinear substrate may be moved down a single mask in a directionperpendicular to the length of the capillary. As it is moved, thecapillary will encounter linear reticles (translucent regions of themask), one at a time, thereby exposing selected regions within thecapillary or capillary. This can allow bundling of parallel capillariesduring synthesis wherein the capillaries are exposed to thicker linearreticles, simultaneously, for a batch processing mode, or individualcapillaries may be placed on a mask having all of the linear reticleslined up so that the capillary can be stepped down the mask in onedirection. Subsequent capillaries may be stepped down the mask at leastone step behind the previous capillary. This employs an assembly linestructure to the substrate preparation process.

As an example, a standard optimization chip for detecting 36simultaneous mutations using a flat geometry chip and an optimizationtiling strategy, is 44×45 features (1980 probes and blanks), with 36blocks of 40 probes each (1440 probes), plus 15 blanks per block (540blank probes). A capillary format, however, can incorporate the samenumber of test probes in a smaller space. Specifically, in a capillarysubstrate, 36 strings of 40 probes will have only one blank spaceseparating each probe group (35 blank probes), for a total of 1475features.

Finally, linear capillary based substrates can provide the advantage ofreduced volume over flat geometries. In particular, typical capillarysubstrates have a volume in the 1-10 μl range, whereas typical flowcells for synthesizing or screening flat geometry chips have volumes inthe range of 100 μl.

A. Stripping and Rinsing

In order to ensure maximum efficiency and accuracy in synthesizingpolymer arrays, it is generally desirable to provide a clean substratesurface upon which the various reactions are to take place. Accordingly,in some processing embodiments of the present invention, the substrateis stripped to remove any residual dirt, oils or other fluorescentmaterials which may interfere with the synthesis reactions, orsubsequent analytical use of the array.

The process of stripping the substrate typically involves applying,immersing or otherwise contacting the substrate with a strippingsolution. Stripping solutions may be selected from a number ofcommercially available, or readily prepared chemical solutions used forthe removal of dirt and oils, which solutions are well known in the art.Particularly preferred stripping solutions are composed of a mixture ofconcentrated H₂SO₄ and H₂O₂. Such solutions are generally available fromcommercial sources, e.g., Nanostrip™ from Cyantek Corp. After stripping,the substrate is rinsed with water and in preferred aspects, is thencontacted with a solution of NaOH, which results in regeneration of aneven layer of hydroxyl functional groups on the surface of thesubstrate. In this case, the substrate is again rinsed with water,followed by a rinse with HCl to neutralize any remaining base, followedagain by a water rinse. The various stripping and rinsing steps maygenerally be carried out using a spin-rinse-drying apparatus of the typegenerally used in the semiconductor manufacturing industry.

Gas phase cleaning and preparation methods may also be applied to thesubstrate wafers using, e.g., H₂O or O₂ plasma or reactive ion etching(RIE) techniques that are well known in the art.

B. Derivatization

Following cleaning and stripping of the substrate surface, the surfaceis derivatized to provide sites or functional groups on the substratesurface for synthesizing the various polymer sequences on that surface.In particular, derivatization provides reactive functional groups, e.g.,hydroxyl, carboxyl, amino groups or the like, to which the firstmonomers in the polymer sequence may be attached. In preferred aspects,the substrate surface is derivatized using silane in either water orethanol. Preferred silanes include mono- and dihydroxyalkylsilanes,which provide a hydroxyl functional group on the surface of thesubstrate. Also preferred are aminoalkyltrialkoxysilanes which can beused to provide the initial surface modification with a reactive aminefunctional group. Particularly preferred are3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane (“APS”).Derivatization of the substrate using these latter amino silanesprovides a linkage that is stable under synthesis conditions and finaldeprotection conditions (for oligonucleotide synthesis, this linkage istypically a phosphoramidate linkage, as compared to the phosphodiesterlinkage where hydroxyalkylsilanes are used). Additionally, this aminosilane derivatization provides several advantages over derivatizationwith hydroxyalkylsilanes. For example, the aminoalkyltrialkoxysilanesare inexpensive and can be obtained commercially in high purity from avariety of sources, the resulting primary and secondary amine functionalgroups are more reactive nucleophiles than hydroxyl groups, theaminoalkyltrialkoxysilanes are less prone to polymerization duringstorage, and they are sufficiently volatile to allow application in agas phase in a controlled vapor deposition process (See below).

Additionally, silanes can be prepared having protected or “masked”hydroxyl groups and which possess significant volatility. As such, thesesilanes can be readily purified by, e.g., distillation, and can bereadily employed in gas-phase deposition methods of silanating substratesurfaces. After coating these silanes onto the surface of the substrate,the hydroxyl groups may be deprotected with a brief chemical treatment,e.g., dilute acid or base, which will not attack the substrate-silanebond, so that the substrate can then be used for polymer synthesis.Examples of such silanes include acetoxyalkylsilanes, such asacetoxyethyltrichlorosilane, acetoxypropyltrimethoxysilane, which maybedeprotected after application using, e.g., vapor phase ammonia andmethylamine or liquid phase aqueous or ethanolic ammonia andalkylamines. Epoxyalkylsilanes may also be used, such asglycidoxypropyltrimethoxysilane which may be deprotected using, e.g.,vapor phase HCl, trifluoroacetic acid or the like, or liquid phasedilute HCl.

The physical operation of silanation of the substrate generally involvesdipping or otherwise immersing the substrate in the silane solution.Following immersion, the substrate is generally spun as described forthe substrate stripping process, i.e., laterally, to provide a uniformdistribution of the silane solution across the surface of the substrate.This ensures a more even distribution of reactive functional groups onthe surface of the substrate. Following application of the silane layer,the silanated substrate may be baked to polymerize the silanes on thesurface of the substrate and improve the reaction between the silanereagent and the substrate surface. Baking typically takes place attemperatures in the range of from 90° C. to 120° C. with 110° C. beingmost preferred, for a time period of from about 1 minute to about 10minutes, with 5 minutes being preferred.

In alternative aspects, as noted above, the silane solution may becontacted with the surface of the substrate using controlled vapordeposition methods or spray methods. These methods involve thevolatilization or atomization of the silane solution into a gas phase orspray, followed by deposition of the gas phase or spray upon the surfaceof the substrate, usually by ambient exposure of the surface of thesubstrate to the gas phase or spray. Vapor deposition typically resultsin a more even application of the derivatization solution than simplyimmersing the substrate into the solution.

The efficacy of the derivatization process, e.g., the density anduniformity of functional groups on the substrate surface, may generallybe assessed by adding a fluorophore which binds the reactive groups,e.g., a fluorescent phosphoramidite such as Fluoreprime™ from Pharmacia,Corp., Fluoredite™ from Millipore, Corp. or FAM™ from ABI, and lookingat the relative fluorescence across the surface of the substrate.

V. Synthesis

General methods for the solid phase synthesis of a variety of polymertypes have been previously described. Methods of synthesizing arrays oflarge numbers of polymer sequences, including oligonucleotides andpeptides, on a single substrate have also been described. See U.S. Pat.Nos. 5,143,854 and 5,384,261 and Published PCT Application No WO92/10092, each of which is incorporated herein by reference in itsentirety for all purposes.

As described previously, the synthesis of oligonucleotides on thesurface of a substrate may be carried out using light directed methodsas described in., e.g., U.S. Pat. Nos. 5,143,854 and 5,384,261 andPublished PCT Application No WO 92/10092, or mechanical synthesismethods as described in U.S. Pat. No. 5,384,261 and Published PCTApplication No. 93/09668, each of which is incorporated herein byreference. Preferably, synthesis is carried out using light-directedsynthesis methods. In particular, these light-directed orphotolithographic synthesis methods involve a photolysis step and achemistry step. The substrate surface, prepared as described hereincomprises functional groups on its surface. These functional groups areprotected by photolabile protecting groups (“photoprotected”), also asdescribed herein. During the photolysis step, portions of the surface ofthe substrate are exposed to light or other activators to activate thefunctional groups within those portions, i.e., to remove photoprotectinggroups. The substrate is then subjected to a chemistry step in whichchemical monomers that are photoprotected at at least one functionalgroup are then contacted with the surface of the substrate. Thesemonomers bind to the activated portion of the substrate through anunprotected functional group.

Subsequent activation and coupling steps couple monomers to otherpreselected regions, which may overlap with all or part of the firstregion. The activation and coupling sequence at each region on thesubstrate determines the sequence of the polymer synthesized thereon. Inparticular, light is shown through the photolithographic masks which aredesigned and selected to expose and thereby activate a first particularpreselected portion of the substrate. Monomers are then coupled to allor part of this portion of the substrate. The masks used and monomerscoupled in each step can be selected to produce arrays of polymershaving a range of desired sequences, each sequence being coupled to adistinct spatial location on the substrate which location also dictatesthe polymer's sequence. The photolysis steps and chemistry steps arerepeated until the desired sequences have been synthesized upon thesurface of the substrate.

Basic strategy for light directed synthesis of oligonucleotides on aVLSIPS™ Array is outlined in FIG. 1. The surface of a substrate or solidsupport, modified with photosensitive protecting groups (X) isilluminated through a photolithographic mask, yielding reactive hydroxylgroups in the illuminated regions. A selected nucleotide, typically inthe form of a 3′-O-phosphoramidite-activated deoxynucleoside (protectedat the 5′ hydroxyl with a photosensitive protecting group), is thenpresented to the surface and coupling occurs at the sites that wereexposed to light. Following capping and oxidation, the substrate isrinsed and the surface is illuminated through a second mask, to exposeadditional hydroxyl groups for coupling. A second selected nucleotide(e.g., 5′-protected, 3′-O-phosphoramidite-activated deoxynucleoside) ispresented to the surface. The selective deprotection and coupling cyclesare repeated until the desired set of products is obtained. Pease etal., Proc. Natl. Acad. Sci. (1994) 91: 5022-5026. Since photolithographyis used, the process can be readily miniaturized to generate highdensity arrays of oligonucleotide probes. Furthermore, the sequence ofthe oligonucleotides at each site is known. Such photolithographicmethods are also described in U.S. Pat. No. 5,143,854, U.S. Pat. No.5,489,678 and Published PCT Application No. WO 94/10128 each of which isincorporated herein by reference in its entirety for all purposes. Inthe large scale processes of the present invention, it is typicallypreferred to utilize photolithographic synthesis methods.

Using the above described methods, arrays may be prepared having allpolymer sequences of a given length which are composed of a basis set ofmonomers. Such an array of oligonucleotides, made up of the basis set offour nucleotides, for example, would contain up to 4^(n)oligonucleotides on its surface, where n is the desired length of theoligonucleotide probe. For an array of 8mer or 10mer oligonucleotides,such arrays could have upwards of about 65,536 and U.S. Pat. No.1,048,576 different oligonucleotides respectively. Generally, where itis desired to produce arrays having all possible polymers of length n, asimple binary masking strategy can be used, as described in U.S. Pat.No. 5,143,854.

Alternate masking strategies can produce arrays of probes which containa subset of polymer sequences, i.e., polymers having a given subsequenceof monomers, but are systematically substituted at each position witheach member of the basis set of monomers. In the context ofoligonucleotide probes, these alternate synthesis strategies may be usedto lay down or “tile” a range of probes that are complementary to, andspan the length of a given known nucleic acid segment. The tilingstrategy will also include substitution of one or more individualpositions within the sequence of each of the probe groups with eachmember of the basis set of nucleotides. These positions are termed“interogation positions.” By reading the hybridization pattern of thetarget nucleic acid, one can determine if and where any mutations lie inthe sequence, and also determine what the specific mutation is byidentifying which base is contained within the interogation position.Tiling methods and strategies are discussed in substantial detail inU.S. patent application Ser. No. 08/143,312 filed Oct. 26, 1993, andincorporated herein by reference in its entirety for all purposes.

Tiled arrays may be used for a variety of applications, such asidentifying mutations within a known oligonucleotide sequence or“target”. Specifically, the probes on the array will have a subsequencewhich is complementary to a known nucleic acid sequence, but wherein atleast one position in that sequence has been systematically substitutedwith the other three nucleotides.

Use of photolabile protecting groups during polymer synthesis has beenpreviously reported, as described above. Preferred photolabileprotecting groups generally have the following characteristics: theyprevent selected reagents from modifying the group to which they areattached; they are stable to synthesis reaction conditions (that is,they remain attached to the molecule); they are removable underconditions that minimize potential adverse effects upon the structure towhich they are attached; and, once removed, they do not reactappreciably with the surface or surface bound oligomer. In someembodiments, liberated byproducts of the photolysis reaction can berendered unreactive toward the growing oligomer by adding a reagent thatspecifically reacts with the byproduct.

The removal rate of the photolabile protecting groups generally dependsupon the wavelength and intensity of the incident radiation, as well asthe physical and chemical properties of the protecting group itself.Preferred protecting groups are removed at a faster rate and with alower intensity of radiation. Generally, photoprotecting groups thatundergo photolysis at wavelengths in the range from 300 nm toapproximately 450 nm are preferred.

Generally, photolabile or photosensitive protecting groups includeortho-nitrobenzyl and ortho-nitrobenzyloxycarbonyl protecting groups.The use of these protecting groups has been proposed for use inphotolithography for electronic device fabrication (see, e.g.,Reichmanis et al., J. Polymer Sci. Polymer Chem. Ed. (1985) 23: 1-8,incorporated herein by reference for all purposes).

Examples of additional photosensitive protecting groups which may beused in the light directed synthesis methods herein described, include,e.g., 1-pyrenylmethyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl,4-methoxyphenacyloxycarbonyl, 3′-methoxybenzoinyloxycarbonyl,3′,5′-dimethoxybenzoinyl-oxycarbonyl2′,3′-dimethoxybenzoinyl-oxycarbonyl,2′,3′-(methylenedioxy)benzoinyloxycarbonyl,N-(5-bromo-7-nitroindolinyl)carbonyl 3,5-dimethoxybenzyloxycarbonyl, andα-(2-methyleneanthraquinone)oxycarbonyl.

Particularly preferred photolabile protecting groups for protection ofeither the 3′ or 5′-hydroxyl groups of nucleotides or nucleic acidpolymers include the o-nitrobenzyl protecting groups described inPublished PCT Application No. WO 92/10092. These photolabile protectinggroups include, e.g., nitroveratryloxycarbonyl (NVOC), nitropiperonyloxycarbonyl (NPOC), α-methyl-nitroveratryloxycarbonyl (MeNVOC),α-methyl-nitropiperonyloxycarbonyl (MeNPOC), 1-pyrenylmethyloxycarbonyl(PYMOC), and the benzylic forms of each of these (i.e., NV, NP, MeNV,MeNP and PYM, respectively), with MeNPOC being most preferred.

Protection strategies may be optimized for different phosphoramiditenucleosides to enhance synthesis efficiency. Examples of such optimizedsynthesis methods are reported in, e.g., U.S. patent application Ser.No. 08/445,332 filed May 19, 1995. Generally, these optimization methodsinvolve selection of particular protecting groups for protection of theO⁶ group of guanosine, which can markedly improve coupling efficienciesin the synthesis of guanosine containing oligonucleotides. Similarly,selection of the appropriate protecting group for protection of the N²group of guanosine can also result in such an improvement, in absence ofprotection of the O⁶ group. For example, suitable protecting groups forprotection of the N² group, where the O⁶ group is also protected,include, e.g., mono- or diacyl protecting groups, triarylmethylprotecting groups, e.g., DMT and MMT, and amidine type protectinggroups, e.g., N,N-dialkylformamidines. Particularly preferred protectinggroups for the N₂ group include, e.g., DMT, DMF, PAC, Bz and Ibu.

Protection of the O⁶ group will generally be carried out using carbamateprotecting groups such as —C(O)NX₂, where X is alkyl, or aryl; or theprotecting group —CH₂CH₂Y, where Y is an electron withdrawing group suchas cyano, p-nitrophenyl, or alkyl- or aryl-sulfonyl; and aryl protectinggroups. In a particularly preferred embodiment, the O⁶ group isprotected using a diphenylcarbamoyl protecting group (DPC).

Alternatively, improved coupling efficiencies may be achieved byselection of an appropriate protecting group for only the N² group. Forexample, where the N²—PAC protecting group is substituted with an Ibuprotecting group, a substantial improvement in coupling efficiency isseen, even without protection of the O⁶ group.

A variety of modifications can be made to the above-described synthesismethods. For example, in some embodiments, it may be desirable todirectly transfer or add photolabile protecting groups to functionalgroups, e.g., NH₂, OH, SH or the like, on a solid support. For thesemethods, conventional peptide or oligonucleotide monomers or buildingblocks having chemically removable protecting groups are used instead ofmonomers having photoprotected functional groups. In each cycle of thesynthesis procedure, the monomer is coupled to reactive sites on thesubstrate, e.g., sites deprotected in a prior photolysis step. Theprotecting group is then removed using conventional chemical techniquesand replaced with a photolabile protecting group prior to the nextphotolysis step.

A number of reagents will effect this replacement reaction. Generally,these reagents will have the following generic structure:

where R₁ is a photocleavable protecting group and X is a leaving group,i.e., from the parent acid HX. The stronger acids typically correspondto better leaving groups and thus, more reactive acylating agents.

Examples of suitable leaving groups include a number of derivativeshaving a range of properties, e.g., relative reactivity, solubility,etc. These groups generally include simple inorganic ions, i.e.,halides, N₃—, and the like, as well as compounds having the followingstructures:

where R₂ is alkyl, substituted alkyl or aryl, R₃ is hydrogen, alkyl,thioalkyl, aryl; R₄ is an electron withdrawing group such as NO₂,SO₂—R₂, or CN; R₅ is a sterically hindered alkyl or aryl group such asadamantyl, t-butyl and the like; and R₆ is alkyl or aryl substitutedwith electronegative substituents. Examples of these latter leavinggroups include:

Conditions for carrying out this transfer are similar to those used forcoupling reaction in solid phase peptide synthesis, or for the cappingreaction in solid phase oligonucleotide synthesis. The solid phaseamine, hydroxyl or thiol groups are exposed to a solution of theprotecting group coupled to the leaving group, e.g., MeNPOC-X in anon-nucleophilic organic solvent, e.g., DMF, NMP, DCM, THF, ACN, and thelike, in the presence of a base catalysts, such as pyridine,2,6-lutidine, TEA, DIEA and the like. In cases where acylation ofsurface groups is less efficient under these conditions, nucleophiliccatalysts such as DMAP, NMI, HOBT, HOAT and the like, may also beincluded to accelerate the reaction through the in situ generation ofmore reactive acylating agents. This would typically be the case where aderivative is preferred for its longer term stability in solution, butis not sufficiently reactive without the addition of one or more of thecatalysts mentioned above. On automated synthesizers, it is generallypreferable to choose a reagent which can be stored for longer terms as astable solution and then activated with the catalysts only when needed,i.e., in the reactor system flow cell, or just prior to the addition ofthe reagent to the flow cell.

In addition to the protection of amine groups and hydroxyl groups inpeptide and oligonucleotide synthesis, the reagents and methodsdescribed herein may be used to transfer photolabile protecting groupsdirectly to any nucleophilic group, either tethered to a solid supportor in solution.

A. Individual Processing

1. Flow Cell/Reactor System

In one embodiment, the substrate preparation process of the presentinvention combines the photolysis and chemistry steps in a single unitoperation. In this embodiment, the substrate wafer is mounted in a flowcell during both the photolysis and chemistry or monomer addition steps.In particular, the substrate is mounted in a reactor system that allowsfor the photolytic exposure of the synthesis surface of the substrate toactivate the functional groups thereon. Solutions containing chemicalmonomers are then introduced into the reactor system and contacted withthe synthesis surface, where the monomers can bind with the activefunctional groups on the substrate surface. The monomer containingsolution is then removed from the reactor system, and another photolysisstep is performed, exposing and activating different selected regions ofthe substrate surface. This process is repeated until the desiredpolymer arrays are created.

Reactor systems and flow cells that are particularly suited for thecombined photolysis/chemistry process include those described in, e.g.,U.S. Pat. No. 5,424,186, which is incorporated herein by reference inits entirety for all purposes,

A schematic illustration of a device for carrying out the combinedphotolysis/chemistry steps of the individual process, is shown in FIGS.3A and 3B. These figures show a cross-sectional view of alternateembodiments of the reactor system 100. Referring first to FIG. 3B, thedevice includes a flow cell which is made up of a body 102 having acavity 104 disposed in one surface. The cavity generally includes fluidinlets 108 and outlets 110 for flowing fluid into and through thecavity. The cavity may optionally include ridges 106 on the back surfaceof the cavity to aid in mixing the fluids as they are pumped into andthrough the cavity. The substrate 112 is mounted over the cavity wherebythe front surface of the substrate wafer 114 (the surface upon which thearrays are to be synthesized) is in fluid communication with the cavity.The device also includes a fluid delivery system in fluid connectionwith the fluid inlet 108 for delivering selected fluids into the cavityto contact the first surface of the substrate. The fluid delivery systemtypically delivers selected fluids, e.g., monomer containing solutions,index matching fluids, wash solutions, etc., from one or more reagentreservoirs 118, into the cavity via the fluid inlet 108. The deliverysystem typically includes a pump 116 and one or more valves to selectfrom the various reagent reservoirs.

For carrying out the photolysis reactions, the device 100 also typicallyincludes a light source 124, as described above. The light source isshown through a photolithographic mask 128 and is directed at thesubstrate 112. Directing the light source at the substrate may generallybe carried out using, e.g., mirrors 122 and/or lenses 120 and 126.Alternatively, as shown in FIG. 3B, the mask 128 may be placed directlyover the substrate 112, i.e., immediately adjacent to the substrate,thereby obviating the need for intervening lenses.

FIGS. 4A and 4B show different views of schematic illustrations of oneembodiment of the flow cell portion of the device, e.g., the bodysubstrate combination. As shown in FIGS. 4A and 4B, a panel 320 ismounted to the body 102 to form the bottom surface of the cavity 104.Silicone cement or other adhesive may be used to mount the panel andseal the bottom of the cavity. In particularly preferred aspects, panel320 will be a light absorptive material, such as yellow glass, RG1000 nmlong pass filter, or other material which absorbs light at the operatingwavelengths, for eliminating or minimizing reflection of impinginglight. As a result, the burden of filtering stray light at the incidentwavelength during synthesis is significantly lessened. The glass panelalso provides a durable surface for forming the cavity since it isrelatively immune to corrosion in the high salt environments or otherconditions common in DNA synthesis reactions or other chemicalreactions.

The substrate wafer 112 is mated to a surface 300. The first surface 114of wafer comprises the photalabile protecting groups coupled tofunctional groups coupled to the substrate surface, as described above.In some embodiments, vacuum pressure may be used to mate the wafer tothe surface 300. In such embodiments, a groove 304, which may be about 2mm deep and 2 mm wide, is formed on surface 300. The groove communicateswith an opening 303 that is connected to a vacuum source, e.g., a pump.The vacuum source creates a vacuum in the groove and causes thesubstrate wafer to adhere to surface 300.

A groove 310 may be formed on surface 300 for seating a gasket 311therein. The gasket ensures that the cavity is sealed when the wafer ismated to the flow cell. Alignment pins 315 may be optionally provided onsurface 300 to properly align the substrate wafer on the flow cell.

Inlet port 307 and outlet port 306 are provided for introducing fluidsinto and flowing fluids out of the cavity. The flow cell provides anopening 301 in which a flow tube 340 is passed through for coupling toinlet port 307. Likewise, a flow tube 341 is passed through opening 302for coupling with outlet port 306. Fittings 345 are employed to maintainthe flow tubes in position. Openings 301 and 302 advantageously positionthe flow tubes so that the flow cell can easily and conveniently bemounted on the synthesis system.

A pump, which is connected to one of the flow tubes, circulates aselected fluid into the cavity and out through the outlet port forrecirculation or disposal. The selected fluids may include, e.g.,monomer containing solutions, index matching fluids, wash solutions orthe like. Although described in terms of a pump, a variety ofpressurized delivery systems may be used to deliver fluids to thecavity. Examples of these alternate systems utilize argon gas tocirculate the selected fluid into and through the cavity.Simultaneously, the flow of argon gas may be regulated to create bubblesfor agitating the fluid as it is circulated through the system.Agitation is used to mix the fluid contents in order to improve theuniformity and/or yield of the reactions.

As shown, inlet and outlet ports 306 and 307, respectively, are locatedat opposite ends of the panel. This configuration improves fluidcirculation and regulation of bubble formation in the cavity. In oneembodiment, the outlet and inlet are located at the top and bottom endsof the cavity, respectively, when the flow cell is mounted vertically onthe synthesizer. Locating the outlet and inlet at the highest and lowestpositions in the cavity, respectively, facilitates the removal ofbubbles from the cavity.

In some embodiments, the flow cell may be configured with a temperaturecontrol system to permit the synthesis reactions to be conducted underoptimal temperature conditions. Examples of temperature control systemsinclude refrigerated or heated baths, refrigerated air circulatingdevices, resistance heaters, thermoelectric peltier devices and thelike.

In some instances, it may be desirable to maintain the volume of theflow cell cavity as small as possible so as to more accurately controlreaction parameters, such as temperature or concentration of chemicals.In addition to the benefits of improved control, smaller cavity volumesmay reduce waste, as a smaller volume requires a smaller amount ofmaterial to carry out the reaction.

For particularly small cavity volumes, a difficulty may arise wherebubbles in the reaction fluids can become trapped in the cavity, whichmay result in incomplete exposure of the substrate surface to thereaction fluid. In particular, when a fluid fills into a very shallowchannel or slit, it will tend to fill the shallowest areas first, due torelatively strong capillary forces in those areas. If the channel is tooshallow, inconsistency and non-flatness of the substrate which resultsin uneven capillary forces, will lead to an uneven fluid front duringfilling. As the liquid front loses its even shape, liquid may surroundair or gas pockets to produce trapped bubbles. Accordingly, whereparticularly small cavity volumes are desired, a flow cell may beemployed wherein the top and bottom surfaces of the flow cell arenonparallel, being narrower at the inlet of the flow cell, and growingwider toward the outlet. Uniform filling of the flow cell ensures thatthe fluid front maintains a straight shape, thereby minimizing thepotential of having bubbles trapped between the surfaces.

A schematic illustration of one embodiment of an integrated reactorsystem is shown in FIG. 4C. The device includes an automated peptidesynthesizer 401. The automated peptide synthesizer is a device whichflows selected reagents through a flow cell 402 under the direction of acomputer 404. In a preferred embodiment the synthesizer is an ABIPeptide Synthesizer, model no. 431A. The computer may be selected from awide variety of computers or discrete logic including, for example, anIBM PC-AT or similar computer linked with appropriate internal controlsystems in the peptide synthesizer. The PC is provided with signals fromthe ABI computer indicative of, for example, the beginning of aphotolysis cycle. One can also modify the synthesizer with a board thatlinks the contacts of relays in the computer in parallel with theswitches to the keyboard of the control panel of the synthesizer toeliminate some of the keystrokes that would otherwise be required tooperate the synthesizer.

Substrate 406 is mounted on the flow cell, forming a cavity between thesubstrate and the flow cell. Selected reagents flow through this cavityfrom the peptide synthesizer at selected times, forming an array ofpeptides on the face of the substrate in the cavity. Mounted above thesubstrate, and preferably in contact with the substrate is a mask 408.Mask 408 is transparent in selected regions to a selected wavelength oflight and is opaque in other regions to the selected wavelength oflight. The mask is illuminated with a light source 410 such as a UVlight source. In one specific embodiment the light source 410 is a modelNo. 82420 made by Oriel. The mask is held and translated by an x-ytranslation stage 412. Translation stages may be obtained commerciallyfrom, e.g., Newport Corp. The computer coordinates the action of thepeptide synthesizer, translation stage, and light source. Of course, theinvention may be used in some embodiments with translation of thesubstrate instead of the mask.

2. Photolysis Step

As described above, photolithographic methods are used to activateselected regions on the surface of the substrate. Specifically,functional groups on the surface of the substrate or present on growingpolymers on the surface of the substrate, are protected with photolabileprotecting groups. Activation of selected regions of the substrate iscarried out by exposing selected regions of the substrate surface toactivation radiation, e.g., light within the effective wavelength range,as described previously. Selective exposure is typically carried out byshining a light source through a photolithographic mask. Alternatemethods of exposing selected regions may also be used, e.g., fiberopticfaceplates, etc. For the individual process methods, e.g., theintegrated photolysis/chemistry process, the substrate is mounted in thereactor system or flow cell such that the synthesis surface of thesubstrate is facing the cavity and away from the light source. As thelight source is shown on the surface opposite that upon which thephotoprotective groups are provided, this method of exposure is termed“backside” photolysis.

Because the individual feature sizes on the surface of the substrateprepared according to the processes described herein can typically rangeas low as 1-10 μm on a side, the effects of reflected or refracted lightat the surface of the substrate can have significant effects upon theability to expose and activate features of this size. One method ofreducing the occurrence of reflected light is to incorporate a lightabsorptive material as the back surface of the flow cell, as describedabove. Refraction of the light as it enters the flow cell, i.e., crossesthe substrate/flow cell interface, through the back surface of thesubstrate can also result in a loss in feature resolution at thesynthesis surface of the substrate resulting from refraction andreflection. To alleviate this problem, during the photolysis step, it isgenerally desirable to fill the flow cell with an index matching fluid(“IMF”) to match the refractive index of the substrate, thereby reducingrefraction of the incident light and the associated losses in featureresolution. The index matching fluid will typically have a refractiveindex that is close to that of the substrate. Typically, the refractiveindex of the IMF will be within about 10% that of the substrate, andpreferably within about 5% of the refractive index of the substrate.Refraction of the light entering the flow cell, as it contacts theinterface between the substrate and the IMF is thereby reduced. Wheresynthesis is being carried out on, e.g., a silica substrate, aparticularly preferred IMF is dioxane which has a refractive indexroughly equivalent to the silica substrate.

The light source used for photolysis is selected to provide a wavelengthof light that is photolytic to the particular protecting groups used,but which will not damage the forming polymer sequences. Typically, alight source which produces light in the UV range of the spectrum willbe used. For example, in oligonucleotide synthesis, the light sourcetypically provides light having a wavelength above 340 nm, to effectphotolysis of the photolabile protecting groups without damaging theforming oligonucleotides. This light source is generally provided by aHg-Arc lamp employing a 340 nm cut-off filter (i.e., passing lighthaving a wavelength greater than 340-350 nm). Typical photolysisexposures are carried out at from about 6 to about 10 times the exposedhalf-life of the protecting group used, with from 8-10 times thehalf-life being preferred. For example, MeNPOC, a preferred photolabileprotecting group, has an exposed half-life of approximately 6 seconds,which translates to an exposure time of approximately 36 to 60 seconds.

Photolithographic masks used during the photolysis step typicallyinclude transparent regions and opaque regions, for exposing onlyselected portions of the substrate during a given photolysis step.Typically, the masks are fabricated from glass that has been coated witha light-reflective or absorptive material, e.g., a chrome layer. Thelight-reflective or absorptive layer is etched to provide thetransparent regions of the mask. These transparent regions correspond tothe regions to be exposed on the surface of the substrate when light isshown through the mask.

In general, it is desirable to produce arrays with smaller featuresizes, allowing the incorporation of larger amounts of information in asmaller substrate area, allowing interogation of larger samples, moredefinitive results from an interogation and greater possibility ofminiaturization. Alternatively, by reducing feature size, one can obtaina larger number of arrays, each having a given number of features, froma single substrate wafer. The result is substantially higher productyields for a given process. This technique, generally referred to as“die shrinking” is commonly used in the semiconductor industry toenhance product outputs or to reduce chip sizes following a over-sizedtest run of a manufacturing process.

In seeking to reduce feature size, it is important to maximize thecontrast between the regions of the substrate exposed to light during agiven photolysis step, and those regions which remain dark or are notexposed. By “contrast” is meant the sharpness of the line separating anexposed region and an unexposed region. For example, the gradient ofactivated to nonactivated groups running from an activated or exposedregion to a nonexposed region is a measure of the contrast. Where thegradient is steep, the contrast is high, while a gradual gradientindicates low or poor contrast.

One cause of reduced contrast is “bleed-over” from exposed regions tonon-exposed regions during a particular photolysis step. In certainembodiments, contrast between features is enhanced through the frontside exposure of the substrate. Front side exposure reduces effects ofdiffraction or divergence by allowing the mask to be placed closer tothe synthesis surface. Additionally, and perhaps more importantly,refractive effects from the light passing through the substrate surfaceprior to exposure of the synthesis surface are also reduced oreliminated by front-side exposure. This is discussed in greater detailbelow.

Contrast between features may also be enhanced using a number of othermethods. For example, the level of contrast degradation between tworegions generally increases as a function of the number of differentialexposures or photolysis steps between the two regions, i.e., incidenceswhere one region is exposed while the other is not. The greater thenumber of these incidences, the greater the opportunity for bleed overfrom one region to the other during each step and the lower the level ofcontrast between the two regions regions. Translated into sequenceinformation, it follows that greater numbers of differences betweenpolymers synthesized in adjacent regions on a substrate can result inreduced contrast between the regions. Namely, the greater the number ofdifferences in two polymer sequences, the greater the number ofincidences of a region bearing the first polymer being exposed while theother was not. These effects are termed “edge” effects as they generallyoccur at the outer edges of the feature.

It is thus desirable to minimize these edge effects to enhance contrastin synthesis. Accordingly, in one aspect, the present invention providesa method of enhancing contrast by reducing the number of differentialsynthesis/photolysis steps between adjacent polymer sequence containingregions throughout an array.

One method of edge minimization is to divide the polymers to besequenced into blocks of related polymers, leaving blank lanes betweenthe blocks to prevent bleed-over into other blocks. While this method iseffective in reducing edge effects, it requires the creation of aspecific algorithm for each new tiling strategy. That is, the layout ofeach block in terms of probe location will depend upon the tiledsequence. In one aspect, the present invention provides methods foraligning polymer synthesis steps on an array whereby the number ofdifferential synthesis steps is reduced, and/or the syntheses inadjacent regions optimized for similarity.

The following example illustrates a typical synthesis strategy. Assuminga simple array where a single possible mutation is being explored at thethird position in the sequence TGTATCA. An array of complementary probesmight be as follows: #1 ACATAGT #2 ACTTAGT #3 ACGTAGT #4 ACCTAGTwhere position 3 has been substituted with each of the four nucleotides.In synthesizing this array, monomer addition is typically cycled throughthe four nucleosides in a given preset order, e.g., 1-A, 2-C, 3-G, 4-T.Thus, for the array shown above, the first “A” in each of the sequenceswould be coupled in the first cycle. The second “C” would be coupled inthe second monomer addition cycle. Each of the substituted positionswould then be coupled in their respective cycle, e.g., the “A” in probe#1 would be coupled in the fifth cycle, while the “T”, “G”, and “C”would be coupled in the sixth, seventh, and eighth cycles, respectively.

Up to this point, each probe has been exposed to a minimal number ofdifferential exposures, as described above. However, the monomeraddition steps following the substituted monomer give rise to somedifficulties in this regard. For example, it would be possible to couplethe “T” in the fourth position in probe #1 at the sixth cycle while the“T” in the remaining probes would have to be added at the tenth cycle,because they could not be added before the preceding monomer in thesequence. The remaining synthesis steps for probe 1 would then be out ofsequence with those of the remaining probes, resulting in an increasednumber of differential sequence steps between probe 1 and the remainingprobes. By aligning the addition of the “T” monomer in probe #1 withthat of the remaining probes, the number of differential synthesis stepsis minimized. Specifically, by waiting until the tenth cycle to add the“T” in probe #1, the number of differential exposures between the probesis minimized to only that number necessary to incorporate the variousmutations or substitutions.

The methods described herein utilize a generalized synthesis method foraligning synthesis steps to accomplish the above-described goal. Thesegeneralized methods can be followed regardless of the particular tilingstrategy used or targeted sequence.

In particular, the methods described herein, identify each probe by ageneric structure which is effectively independent of the actualtargeted sequence. This generic description of a probe sequence istermed an “image”, a collection of polymer sequences is termed a“picture”, and a local translation, e.g., in a larger targeted sequence,is termed a “frame”. The entire picture and frame structure is termed a“collage”.

Each position in the probe is designated by the position number in theframe, or targeted sequence segment, followed by a number that indicatesthe rotation from the wild type monomer, with the wild type monomerbeing “0”. By rotation is meant the number of cycles required to go fromthe wild type monomer to the substituted monomer in the addition cycle(note that a “0” and a “4” are the same monomer in terms ofnucleotides). For example, if a given wild type sequence has an “A” in agiven position, a substitution to a “G” would be identified by arotation of “3”, assuming a monomer addition or synthesis cycle of A, C,T, G.

In terms of the above example, probe #1, being the same as the wild typetarget as also described above, would be identified as:

-   -   #1<1,0><2,0><3,0><4,0><5,0><6,0><7,0>

where each position is not rotated from the wild type, or is“unmodified.” The remaining sequences would be identified as: #2<1,0><2,0><3,1><4,0><5,0><6,0><7,0> #3<1,0><2,0><3,2><4,0><5,0><6,0><7,0> #4<1,0><2,0><3,3><4,0><5,0><6,0><7,0>indicating a rotation in the third position for each of the nucleosidemonomers.

Sequence positions which are in the same layer are aligned to be addedin the same synthesis cycle. The “depth” of the sequence or the “layer”in which a given monomer is found, are determined by counting eachoccurrence where an unmodified base follows a modified base. Eachsequence has a depth of at least one. For example, the sequence “X”indicated by the <1,1><2,0><3,0> has a depth of 2, where <2,0> and <3,0>are in the second layer. Similarly, the sequence “Y” identified as<1,0><2,1><3,0> has a depth of two where <1,0> is in the first layer and<3,0> is in the second layer. Aligning these two sequences, it can beseen that the monomer <3,0> in sequences X and Y may be aligned as itexists in the same layer.

In contrast, the sequence “Z”<1,0><2,0><3,1> has a depth of one with<1,0><2,0> in the first layer. Thus, the position <2,0> in the sequenceX would not be aligned with the same position in sequence Z as theyexist in different layers.

A specific example of the collage method is illustrated using thefollowing sequence/tiling strategy. A targeted sequence is complementaryto the sequence CTTA. Thus, written in the above-described genericstyle, the wild type sequence would be designated <1,0><2,0><3,0><4,0>.Assuming a simplified tiling strategy where each position was to besubstituted with a monomer rotated one from the wild type, the arraywould have the generic description: #1 <1,1><2,0><3,0><4,0> #2<1,0><2,1><3,0><4,0> #3 <1,0><2,0><3,1><4,0> #4 <1,0><2,0><3,0><4,1>

which would correspond to the sequences: #1 G T T A #2 C A T A #3 C T AA #4 C T T C

The assignment of bases of each layer to a particulr cycle is termed a“frame.” For example, the frame for the above synthesis would be asfollows: Layer 1 Layer 2 <1,0> = 2

<2,0> = 4 <2,0> = 8 <3,0> = 8 <3,0> = 12 <4,0> = 13

Once monomers in the same layer are aligned, the synthesis is carriedout with the following aligned cycle assignments: CycleA  C  G  T  A  C  G  T  A  C  G  T  A  C  G  T                        Layer 2 #1       3             8          12 13#2    2       5                    12 13 #3   2     4 5                       13            #4   2     4            8     10                     Layer 1

The bases in the first layer are assigned the cycles closest to thestart of the synthesis. The modified bases (between the layers) areassigned the next available cycles. The second layer is assigned a setof cycles as close as possible to the start of synthesis consistent withthe bases already assigned (i.e., without altering the base ordering ofany of the probes). Subsequent layers are assigned in a similar manner.This method allows maximum alignment of synthesis cycles throughout theframe being synthesized, while minimizing the total length of synthesis(e.g., number of steps).

Another method of minimizing bleed-over in the photolysis steps is toreduce the size of the transmissive or translucent portion of the mask,thus preventing unintentional exposure of adjoining regions caused bydiffraction of the light shown through the mask. In particular, typicalphotolysis steps can have a duration of up to 8 to 10 times thehalf-life of the photodeprotection reaction. Thus, photoprotection canbe up to 50% complete where the light intensity is only 12% of optimallevels, i.e., the level required for complete or near completephotodeprotection. Typically, such intensity levels may be reached welloutside the feature boundary as defined by the transmissive portion ofthe mask.

Reducing the size of the transmissive portion of the mask allowsdiffraction, scattering and divergence at the edges of each featurewithout that diffraction interfering with neighboring features. Thus,the region of incomplete photolysis can be centered on the desiredboundary between features. As a result, the total area of the chip thatis compromised in a multi-step synthesis is minimized because bleed-overeffects from each region are centered in the boundary rather than wellinto the neighboring feature. Accordingly, in one aspect of the presentinvention provides a method of minimizing bleed-over in adjoining cellsby reducing the size of the transmissive portion of the mask, such thatthe zone of divergent light shown through the mask is centered on thedesired feature border. As an example, a mask exposing a rectangularfeature can be reduced by, e.g., 20 μm in each dimension, thus allowinggreater homogeneity at the edges of 100 μm features. In preferredaspects, the translucent region of the mask will be from about 2% toabout 25% smaller in each dimension of the size of the region which isto be exposed. In more preferred aspects, the translucent portion of themask will be from about 10% to about 25% smaller in each dimension.

3. Chemistry Step

Following each photolysis step, a monomer building block is introducedor contacted with the synthesis surface of the substrate. Typically, theadded monomer includes a single active functional group, for example, inthe case of oligonucleotide synthesis, a 3′-hydroxyl group. Theremaining functional group that is involved in linking the monomerwithin the polymer sequence, e.g., the 5′-hydroxyl group of anucleotide, is generally photoprotected. The monomers then bind to thereactive moieties on the surface of the substrate, activated during thepreceding photolysis step, or at the termini of linker molecules orpolymers being synthesized on the substrate.

Typically, the chemistry step involves solid phase polymer synthesismethods that are well known in the art. For example, detaileddescriptions of the procedures for solid phase synthesis ofoligonucleotides by phosphoramidite, phosphite-triester,phosphotriester, and H-phosphonate chemistries are widely available.See, for example, Itakura, U.S. Pat. No. 4,401,796; Caruthers et al.,U.S. Pat. Nos. 4,458,066 and 4,500,707; Beaucage et al., TetrahedronLett., 22: 1859-1862 (1981); Matteucci et al., J. Amer. Chem. Soc., 103:3185-3191 (1981); Caruthers et al., Genetic Engineering, 4: 1-17 (1982);Jones, chapter 2, Atkinson et al., chapter 3, and Sproat et al., chapter4, in Gait, ed. Oligonucleotide Synthesis: A Practical Approach, IRLPress, Washington D.C. (1984); Froehler et al., Tetrahedron Lett., 27:469-472 (1986); Froehler et al., Nucleic Acids Res., 14: 5399-5407(1986); Sinha et al. Tetrahedron Lett., 24: 5843-5846 (1983); and Sinhaet al., Nucl. Acids Res., 12: 4539-4557 (1984).

In operation, during the chemistry/monomer addition step, the IMF isremoved from the flow cell through an outlet port. The flow cell is thenrinsed, e.g., with water and/or acetonitrile. Following rinsing, asolution containing an appropriately protected monomer to be coupled inthe particular synthesis step is added. For example, where the synthesisis of oligonucleotide probe arrays, being synthesized in the 3′ to 5′direction, a solution containing a 3′-O-activated phosphoramiditenucleoside, photoprotected at the 5′ hydroxyl is introduced into theflow cell for coupling to the photoactivated regions of the substrate.Typically, the phosphoramidite nucleoside is present in the monomersolution at a concentration of from 1 mM to about 100 mM, with 10 mMnucleoside concentrations being preferred. Typically, the couplingreaction takes from 30 seconds to 5 minutes and preferably takes about1.5 minutes.

Following coupling, the monomer solution is removed from the flow cell,the substrate is again rinsed, and the IMF is reintroduced into the flowcell for another photolysis step. The photolysis and chemistry steps arerepeated until the substrate has the desired arrays of polymerssynthesized on its surface.

For each photolysis/chemistry cycle, it will generally be desirable tomaximize coupling efficiencies in order to maximize probe densities onthe arrays. Coupling efficiencies may be improved through a number ofmethods. For example, coupling efficiency may be increased by increasingthe lipophilicity of the building blocks used in synthesis. Withoutbeing bound to any theory of operation, it is believed that suchlipophilic building blocks have enhanced interaction at the surface ofthe preferred crystalline substrates. The lipophilicity of the buildingblocks may generally be enhanced using a number of strategies. Inoligonucleotide synthesis, for example, the lipophilicity of the nucleicacid monomers may be increased in a number of ways. For example, one canincrease the lipophilicity of the nucleoside itself, the phosphoramiditegroup, or the protecting group used in synthesis.

Modification of the nucleoside to increase its lipophilicity generallyinvolves specific modification of the nucleobases. For example,deoxyguanosine (dG) may be alkylated on the exocyclic amino group (N2)with DMT-C₁, after in situ protection of both hydroxyl groups astrimethylsilylethers (See, FIG. 5A). Liberation of the free DMTprotected nucleoside is achieved by base catalyzed methanolosis of thedi-TMS ether. Following standard procedures, two further steps are usedresulting in the formation of 5′-MeNPOC-dG-phosphoramidites. The DMTgroup is used because the normally used 5′-DMT-phosphoramidites showhigh coupling efficiencies on silica substrate surfaces and because ofthe ease of synthesis for the overall compound. The use of acid labileprotecting groups on the exocyclic amino groups of dG allows continuedprotection of the group throughout light-directed synthesis. Similarprotection can be used for other nucleosides, e.g., deoxycytosine (dC).Protection strategies for nucleobase functional groups, including theexocyclic groups are discussed in U.S. patent application Ser. No.08/445,332 filed May 19, 1995, previously incorporated herein byreference.

A more lipophilic phosphoramidite group may also be used to enhancesynthesis efficiencies. Typical phosphoramidite synthesis utilizes acyanoethyl-phosphoramidite. However, lipophilicity may be increasedthrough the use of, e.g., an Fmoc-phosphoramidite group. Synthesis ofFmoc-phosphoramidites is shown in FIG. 5B. Typically, aphosphorus-trichloride is reacted with four equivalents ofdiisopropylamine, which leads to the formation of the correspondingmonochloro-bisamino derivative. This compound reacts with theFmoc-alcohol to generate the appropriate phosphatidylating agent.

As with the phosphoramidite group, the photolabile protecting groups mayalso be made more lipophilic. For example, a lipophilic substituent,e.g., benzyl, naphthyl, and the like, may be introduced as analkylhalide, through α-akylation of a nitroketone, as shown in FIG. 5C.Following well known synthesis techniques, one generates thechloroformate needed to introduce the photoactive lipophilic group tothe 5′ position of a deoxyribonucleoside.

B. Batch Processing

In a second embodiment of the substrate preparation process, each of thephotolysis and chemistry steps involved in the synthesis operation areprovided as separate unit operations. This method provides advantages ofefficiency and higher feature resolution over the single unit operationprocess. In particular, the separation of the photolysis and chemistrysteps allows photolysis to be carried out outside of the confines of theflow cell. This permits application of the light directly to thesynthesis surface, i.e., without first passing through the substrate.This “front-side” exposure allows for greater definition at the edges ofthe exposed regions (also termed “features”) by eliminating therefractive influence of the substrate and allowing placement of the maskcloser to the synthesis surface. A comparison illustrating the improvedresolution of front-side synthesis is shown in FIGS. 8A-8D.

In addition to the benefits of front side exposure, the batch methodprovides advantages in the surface area of a substrate wafer that may beused in synthesizing arrays. In particular, by combiningphotolysis/chemistry aspects in the individual process methods, theoperation of mounting the substrate wafer on the flow call can result inless than the entire surface of the substrate wafer being used forsynthesis. In particular, where the substrate wafer is used to form onewall of the flow cell, as is typically the case in these combinedmethods, engineering constraints involved in mounting of the flow cellcan result in a reduction in the available substrate surface area. Thisis particularly the case where a vacuum chuck system is used to mountthe substrate on the flow cell, where the vacuum chuck system requires acertain amount of surface area to hold the substrate on the flow cellwith sufficient force.

In batch mode operation, the chemistry step is generally carried out byimmersing the entire substrate wafer in the monomer solution, thusallowing synthesis over most if not all of the substrate wafer'ssynthesis surface. This results in a higher chip yield per substratewafer than in the individual processing methods. Additionally, as thechemistry steps are generally the time limiting steps in the synthesisprocess, monomer addition by immersion permits monomer addition tomultiple substrates at a given time, while more substrates areundergoing the photolysis steps.

For example, where synthesis is performed in the individual processingoperation, as described above, the engineering constraints in vacuummounting a substrate to a flow cell can result in a significant decreasein the size of a synthesis area on the substrate wafer. For example, inone process, a substrate wafer having dimensions of 5″×5″ has only2.5″×2.5″ available as a synthesis surface, which when separated intochips of typical dimensions (e.g., 1.28 cm×1.28 cm) typically results in16 potential chips per wafer. The same sized wafer, when subjected tothe batch mode synthesis can have a synthesis area of about 4.3″×4.3″,which can produce approximately 49 chips per wafer.

In general, a number of substrate wafers is subjected to the photolysisstep. Following photolysis, the number of wafers is placed in a rack or“boat” for transport to the station which performs the chemistry steps,whereupon one or more chemistry steps are performed on the wafers,simultaneously. The wafers are then returned to the boat and transportedback to the station for further photolysis. Typically, the boat is arack that is capable of carrying several wafers at a time and is alsocompatible with automated systems, e.g., robotics, so that the wafersmay be loaded into the boat, transported and placed into the chemistrystation, and following monomer addition returned to the boat and thephotolysis station, all through the use of automated systems.

Initial substrate preparation is the same for batch processing asdescribed in the individual processing methods, above. However, beyondthis initial substrate preparation, the two process take divergentpaths. In batch mode processing, the photolysis and chemistry steps areperformed separately. As is described in greater detail below, thephotolysis step is generally performed outside of the flow cell. Thiscan cause some difficulties, as there is no provision of an IMF behindthe substrate to prevent the potentially deleterious effects ofrefraction and reflection of the photolytic light source. In someembodiments, however, the same goal is accomplished by applying acoating layer to the back-side of the substrate, i.e., to thenon-synthesis surface of the substrate. The coating layer is typicallyapplied after the substrate preparation process, but prior toderivatization. This coating is typically selected to perform one ormore of the following functions: (1) match the refractive index of thesubstrate to prevent refraction of light passing through the substratewhich may interfere with the photolysis; and (2) absorb light at thewavelength of light used during photolysis, to prevent back reflectionwhich may also interfere with photolysis.

Typically, suitable coating materials may be selected from a number ofsuitable materials which have a refractive index approximately equal tothat of the substrate and/or absorb light at the appropriate wavelength.In particular, index matching coatings are typically selected to have arefractive index that is within at about 10% that of the substrate, andpreferably within about 5%. Similarly, light absorbing coatings aretypically selected whereby light at the photolytic wavelength isabsorbed, which in preferred aspects is light in the ultraviolet range,e.g., between 280 nm and 400 nm. Light absorbing coatings and indexmatching coatings may be combined to provide combined protection againstrefraction and reflection, or a single coating material may be selectedwhich possesses both of the desired properties.

Preferred polymers will typically be selected to be compatible with thevarious reaction conditions which would be encountered during thesynthesis process, e.g., insoluble in and non-reactive with synthesisreagents, and resistant to the mechanical forces involved in handlingand manipulating the substrate, throughout the synthesis process.Additionally, preferred coating materials are easily removable uponcompletion of the synthesis process, e.g., in the final deprotectionstep or in a final coating removal step.

Examples of suitable coating materials include anti-reflective coatingsthat are well known in the art and generally commercially available,e.g., magnesium fluoride compounds, which are light-absorbing in thedesired wavelength range, polymethylmethacrylate coatings (PMMA), whichhave a refractive index comparable to glass substrates, and polyimidecoatings which are both light-absorbing in the desired wavelength range,and have a refractive index close to that of a glass substrate.Polyimide coatings are most preferred.

Application of the coating materials may be carried out by a variety ofmethods, including, e.g., vapor deposition, spray application, and thelike. In preferred aspects, the coating solution will be applied to thesubstrate using a spin-coating method. Typically, this involves spinningthe substrate during deposition of the coating solution on the substratesurface that is to be coated. The spinning substrate results inspreading of the coating solution radially outward on the surface of thesubstrate.

Application of the coating material using the spin-coating processusually employs a two-speed spinning of the substrate. The applicationof the coating material to the surface of the substrate and initialspreading of the coating solution are usually carried out at lowrotational speeds and for relatively short duration. For example, toapply 1 ml of a 12% solids w/v polymer coating solution to a 4.3″×4.3″substrate, initial spreading is carried out at 500 r.p.m. for 10seconds. Elimination of excess polymer solution and evening of thepolymer layer are carried out at higher rotational speeds and forsubstantially longer durations. For example in the application describedabove, the second spinning step is carried out at approximately 3000r.p.m. for 30 seconds. It will be understood by those of skill in theart, that the above described parameters for spin-coating can be variedwithin the scope of the present invention. For example, where higherconcentration (w/v) polymer solutions are used, it may be desirable toincrease one or both rotational speeds, as well as the time at a givenspeed. Similarly, where the polymer concentration in the polymersolution is reduced, lower speeds and shorter spin times may be used.

Following application, the polymer coating is then cured on the surfaceof the substrate. Curing is typically carried out by heating the coatedsubstrate. In preferred processes, the curing process involves atwo-step heating process. The first step involves a “soft-bake” heatingof the coated substrate to initially cure the polymer coating. Thissoft-bake step typically takes place at relatively low temperatures forrelatively short periods, i.e., 85° C. for 5 minutes. The second step ofthe curing process is a final curing of the polymer coating which istypically carried out at higher temperatures for longer periods, i.e.,220-360° C., for approximately 60 minutes. In preferred aspects, apolymer coating applied to the back side of the substrate will be fromabout 1 to about 50 μm thick, and more preferably, from about 5 to about20 μm thick, with polymer coating of about 10 μm thick being mostpreferred.

The back-side coated substrate is then subjected to derivitization,rinsing and baking, according to the above described methods.

As described previously, the steps of photolysis and monomer addition inthe batch mode aspects of the present invention are performed inseparate unit operations. Separation of photolysis and chemistry stepsallows a more simplified design for a photolyzing apparatus.Specifically, the apparatus need not employ a flow cell. Additionally,the apparatus does not need to employ a particular orientation to allowbetter filling of the flow cell. Accordingly, the apparatus willtypically incorporate one or more mounting frames to immobilize thesubstrate and mask during photolysis, as well as a light source. Thedevice may also include focusing optics, mirrors and the like fordirecting the light source through the mask and at the synthesis surfaceof the substrate. As described above, the substrate is also placed inthe device such that the light from the light source impacts thesynthesis surface of the substrate before passing through the substrate.As noted above, this is termed “front-side” exposure.

Typically a photolysis step requires far less time than a typicalchemistry step, e.g., 60 seconds as compared to 10 minutes. Thus, in theindividual processing mode where the photolysis and chemistry steps arecombined, the photolysis machinery sits idle for long periods of timeduring the chemistry step. Batch mode operation, on the other hand,allows numerous substrates to be photolyzed while others are undergoinga particular chemistry step. For example, a number of substrate wafersmay be exposed for a given photolysis step. Following photolysis, theseveral substrate wafers may be transferred to a number of reactionchambers for the monomer addition step. While monomer addition is beingcarried out, additional substrate wafers may be undergoing photolysis.

FIG. 6A schematically illustrates a bank of reaction chambers forcarrying simultaneous monomer addition steps on a number of separatesubstrates in parallel. As shown, the bank of reaction chambers isconfigured to simultaneously perform identical synthesis steps in eachof the several reaction chambers. Each reaction chamber 602 is equippedwith a fluid inlet 604 and outlet 606 for flowing various fluids intoand through the reaction chamber. The fluid inlet of each chamber isgenerally fluidly connected to a manifold 608 which connects all of thereaction chambers, in parallel, to a single valve assembly 610.Typically, rotator valves are preferred for this aspect of theapparatus. The valve assembly allows the manifold to be fluidlyconnected to one of a plurality of reagent vessels 612-622. Alsoincluded is a pump 624 for delivering the various reagents to thereaction chamber. Although primarily described as performing the samesynthesis steps in parallel, the bank of reaction chambers could also bereadily modified to carry out to perform multiple independent chemistrysteps. The outlet ports 606 from the reaction chambers 602 are typicallyfluidly connected to a waste vessel (not shown).

FIG. 6B shows a schematic representation of a single reaction chamberfor performing the chemistry steps of the batch process, e.g., monomeraddition. As shown, the reaction chamber employs a “clam-shell” designwherein the substrate is enclosed in the reaction chamber 602 when thedoor 652 is closed against the body 654 of the apparatus. Moreparticularly, the substrate wafer 660 is mounted on the chamber door andheld in place, e.g., by a vacuum chuck shown as vacuum groove 670. Whenthe door 652 is closed, the substrate wafer 668 is placed into thereactor cavity 656 on the body of the device. The reactor cavity issurrounded by a gasket 658, which provides the seal for the reactionchamber when the door is closed. Upon closing the door, the substratewater is pressed against the gasket and the pressure of this contactseals the reaction chamber. The reaction chamber includes a fluid inlet604 and a fluid outlet 606, for flowing monomer solutions into and outof the reaction chamber.

The apparatus may also include latches 666, for locking the reactionchamber in a sealed state. Once sealed, reagents are delivered into thereaction chamber through fluid inlet 662 and out of the reaction chamberthrough fluid outlet 664. The reaction chamber also typically includes atemperature control element for maintaining the reaction chamber at theoptimal synthesis temperature. As shown, the reaction chamber includesautomatic alignment pins 672, e.g., solenoid or servo operated, foraligning a substrate wafer on the vacuum groove 670.

Following a monomer addition step, the substrate wafers are eachsubjected to a further photolysis step. The process may generally betimed whereby during a particular chemistry step, a new series of wafersis being subjected to a photolysis step. This dramatically increases thethroughput of the process.

Following overall synthesis of the desired polymers on the substratewafers, permanent protecting groups, e.g., those which were not removedduring each synthesis step, typically remain on nucleobases and thephosphate backbone of synthetic oligonucleotides. Removal of theseprotecting groups is usually accomplished with a concentrated solutionof aqueous ammonium hydroxide. While this method is effective for theremoval of the protecting groups, these conditions can also cleave thesynthetic oligomers from the support (usually porous silica particles)by hydrolyzing an ester linkage between the oligo and a functionalizedsilane derivative that is bonded to the support. In VLSIPSoligonucieotide arrays, it is desirable to preserve the linkageconnecting the oligonucleotides to the glass after the finaldeprotection step. For this reason, synthesis is carried out directly onglass which is derivatized with a hydroxyalkyl-trialkoxysilane (e.g.,bis(hydroxyethyl)aminopropylsilane). However, these supports are notcompletely stable to the alkaline hydrolysis conditions used fordeprotection. Depending upon the duration, substrates left in aqueousammonia for protracted periods can suffer a loss of probes due tohydroxide ion attack on the silane bonded phase.

Accordingly, in preferred embodiments, final deprotection of the polymersequences is carried out using anhydrous organic amines. In particular,primary and secondary alkylamines are used to effect final deprotection.The alkylamines may be used undiluted or in a solution of an organicsolvent, e.g. ethanol, acetonitrile, or the like. Typically, thesolution of alkyl amine will be at least about 50% alkylamine (v/v). Avariety of primary and secondary amines are suitable for use indeprotection, including ammonia, simple low molecular weight(C₁₋₄)alkylamines, and substituted alkylamines, such as ethanolamine andethylenediamine. More volatile amines are preferred where removal of thedeprotection agent is to be carried out by evaporation, whereas the lessvolatile amines are preferred in instances where it is desirable tomaintain containment of the deprotection agent and where the solutionsare to be used in repeated deprotections. Solutions of ethanolamine orethylenediamine in ethanol have been used in deprotecting syntheticoligonucleotides in solution. See, Barnett, et al., Tet. Lett. (1981)22: 991-994, Polushin, et al, (1991) N.A.R. Symp. Ser. No. 24: 49-50 andHogrefe, et al. N.A.R. (1993) 21: 2031-2038.

Depending upon the protecting groups to be removed, the time requiredfor complete deprotection in these solutions ranges from several minutesfor “fast” base-protecting groups, e.g. PAC or DMF-protected A, C or Gand Ibu-protected C, to several hours for the standard protectinggroups, e.g. benzoyl-protected A, C or G and Ibu-protected G. Bycomparison, even the fast protecting groups require 4-8 hours forcomplete removal in aqueous ammonia. During this time, a significantpercentage (e.g., 20-80%) of probes are cleaved from a glass substratethrough hydrolytic cleavage of the silane layer, whereas after 48 hoursof exposure to 50% ethanolic ethylenediamine solution, 95% of the probesremain on the substrate.

VI. Assembly of Probe Array Cartridges

Following synthesis, final deprotection and other finishing steps, e.g.polymer coat removal where necessary, the substrate wafer is assembledfor use as individual substrate segments. Assembly typically employs thesteps of separating the substrate wafer into individual substratesegments, and inserting or attaching these individual segments to ahousing which includes a reaction chamber in fluid communication withthe front surface of the substrate segment, e.g., the surface having thepolymers synthesized thereon.

Methods of separating and packaging substrate wafers are described insubstantial detail in Published PCT Application No. 95/33846, which ishereby incorporated herein by reference in its entirety for allpurposes.

Typically, the arrays are synthesized on the substrate wafer in a gridpattern, with each array being separated from each other array by ablank region where no compounds have been synthesized. These separatingregions are termed “streets”. The wafer typically includes a number ofalignment marks located in these streets. These marks serve a number ofpurposes, including aligning the masks during synthesis of the arrays asdescribed above, separation of the wafer into individual chips andplacement of each chip into its respective housing for subsequent use,which are both described in greater detail below. An illustration of awafer including these alignment marks is shown in FIG. 7. As shown,substrate wafer 700 includes individual arrays 710 separated by streets720 and includes alignment marks 730.

Generally, the substrate wafer can be separated into a number ofindividual substrates using scribe and break methods that are well knownin the semiconductor manufacturing industry. For example, well knownscribe and break devices may be used for carrying out the separationsteps, e.g., a fully programmable computer controlled scribe and breakdevices, such as a DX-III Scriber-Breaker manufactured by DynatexInternational™, or the LCD-1 scriber/dicer manufactured by LoomisIndustries. The steps typically involve scribing along the desiredseparation points, e.g., between the individual synthesized arrays onthe substrate wafer surface, followed by application of a breaking forcealong the scribe line. For example, typical scribe and break devicesbreak the wafer by striking the bottom surface of the wafer along thescribe lines with an impulse bar, or utilizing a three point beamsubstrate bending operation. The shock from the impulse bar fracturesthe wafer along the scribe line. Because the majority of force appliedby the impulse bar is dissipated along the scribe line, the device isable to provide high breaking forces without exerting significant forceon the substrate itself, allowing separation of the wafer withoutdamaging the individual chips.

In alternative methods, the wafer may be separated into individualsegments by, e.g., sawing methods, such as those described in U.S. Pat.No. 4,016,855.

Once the wafer is separated into individual segments, these segments maybe assembled in a housing that is suited for the particular analysis forwhich the array will be used. Examples of methods and devices forassembling the substrate segments or arrays in cartridges are describedin, e.g., U.S. patent application Ser. No. 08/485,452, previouslyincorporated by reference. Typically, the housing includes a body havinga cavity disposed within it. The substrate segment is mounted over thecavity on the body such that the front side of the segment, e.g., theside upon which the polymers have been synthesized, is in fluidcommunication with the cavity. The bottom of the cavity may optionallyinclude a light absorptive material, such as a glass filter or carbondye, to prevent impinging light from being scattered or reflected duringimaging by detection systems. This feature improves the signal-to-noiseratio of such systems by significantly reducing the potential imaging ofundesired reflected light.

The cartridge also typically includes fluid inlets and fluid outlets forflowing fluids into and through the cavity. A septum, plug, or otherseal may be employed across the inlets and/or outlets to seal the fluidsin the cavity. The cartridge also typically includes alignmentstructures, e.g., alignment pins, bores, and/or an asymmetrical shape toensure correct insertion and/or alignment of the cartridge in theassembly devices, hybridization stations, and reader devices.

An illustration of one embodiment of the array cartridge is shown inFIG. 8. FIG. 8 shows a top view 802, end view 804, side view 806 andbottom view 808 of the array cartridge 800. The body of the arraycartridge may generally be fabricated from one or more parts or casings810-814 that are made using a number of manufacturing techniques. Inpreferred aspects, the cartridge is fabricated from two or moreinjection molded plastic parts. Injection molding enables the parts tobe formed inexpensively. Also, assembling the cartridge from two partssimplifies the construction of various features, such as the internalchannels for introducing fluids into the cavity. As a result, thecartridges may be manufactured at a relatively low cost.

The top and bottom views of the cartridge include alignment structures,such as alignment holes 816 and 818. As shown, these alignment holes aredisposed through the body of the cartridge, however, those of ordinaryskill will appreciate that other alignment structures, e.g., alignmentpins, etc., would be equally useful. As shown in the bottom view 808,alignment holes 816 and 818 also include an annular bevelled region toassist in insertion of complementary alignment pins on the hybridizationstation.

Referring to the top view 802 of the cartridge 800, cavity 820 includesa flat bottom peripheral portion 822, a bevelled portion 824 extendingfrom the flat bottom peripheral portion, and a flat upper portion 826surrounding the beveled portion. The array includes an outer peripherywhich rests against the flat bottom peripheral portion 822. The bevelledportion aligns the chip onto the flat bottom peripheral portion 822. Asshown, the top casing 814 extends outside the middle and bottom casings,812 and 810, respectively, to provide a nonflush edge 828. The alignmentstructures 816 and 818, as well as the non flush edge 828, ensure properorientation of the cartridge in the hybridization station, as well asother devices used in producing and reading polymer arrays. Surroundingmounting structures 816 and 818 are annular recesses 817 and 819,respectively, which aid in guiding the cartridge onto complementarymounting structures on the various devices.

As shown in the bottom view 808, the cartridge includes inlet and outletports 830 and 834, which include a bevelled annular region 832 and 836surrounding these ports, respectively, to assist with fluid flowtherethrough. Typically, the inlet and outlet ports will include septadisposed across the ports (not shown). Bottom casing 810 also includes acavity 838, located adjacent the array, which cavity may be adapted forreceiving a temperature monitoring and/or controlling device. As shownthe cavity 838 has an annular recessed region 839 surrounding it, toensure that the temperature controller may be inserted with maximumease.

The array cavity 820 is preferably located at a center of the bottomcasing, but may also be at other locations. The cavity may be round,square, rectangular, or any other shape, and orientation. The cavity ispreferably smaller than the surface area of the chip to be placedthereon, and has a volume sufficient to perform hybridization and thelike. In one embodiment, the cavity includes dimensions such as a lengthof about 0.6 inch, a width of about 0.6 inch and a depth of about 0.07inch.

In a preferred embodiment, the bottom casing with selected cavitydimensions may be removed from the middle and top casings, and replacedwith another bottom casing with different cavity dimensions. This allowsa user to attach a chip having a different size or shape by changing thebottom casing, thereby providing ease in using different chip sizes,shapes, and the like. Of course, the size, shape, and orientation of thecavity will depend upon the particular application. The body of thecartridge may generally be fabricated from one or more parts made usinga number of manufacturing techniques. In preferred aspects, thecartridge is fabricated from two or more injection molded plastic parts.Injection molding enables the casings to be formed inexpensively. Also,assembling the cartridge from two parts simplifies the construction ofvarious features, such as the internal channels for introducing fluidsinto the cavity. As a result, the cartridges may be manufactured at arelatively low cost.

The substrate segment may be attached to the body of the cartridge usinga variety of methods. In preferred aspects, the substrate is attachedusing an adhesive. Preferred adhesives are resistant to degradationunder conditions to which the cartridge will be subjected. Inparticularly preferred aspects, an ultraviolet cured adhesive attachesthe substrate segment to the cartridge. Devices and methods forattaching the substrate segment are described in Published PCTApplication No. 95/33846, previously incorporated by reference.Particularly preferred adhesives are commercially available from avariety of commercial sources, including Loctite Corp. and Dymax Corp.

A variety of modifications can be incorporated in the assembly methodsand devices that are generally described herein, and these too areoutlined in greater detail in published PCT Application No. 95/33846.

Upon completion, the cartridged substrate will have a variety of uses.For example, the cartridge can be used in a variety of sequencing byhybridization (“SBH”) methods, sequence checking methods, diagnosticmethods and the like. Arrays which are particularly suited for sequencechecking and SBH methods are described in, e.g. U.S. patent applicationSer. No. 08/505,919, filed Jul. 24, 1995, Ser. No. 08/441,887, filed May16, 1995, Ser. No. 07/972,007, filed Nov. 5, 1992, each of which isincorporated herein by reference in its entirety for all purposes.

Typically, in carrying out these methods, the cartridged substrate ismounted on a hybridization station where it is connected to a fluiddelivery system. The fluid delivery system is connected to the cartridgeby inserting needles into the inlet and outlet ports through the septadisposed therein. In this manner, various fluids are introduced into thecavity for contacting the probes synthesized on the front side of thesubstrate segment, during the hybridization process.

Usually, hybridization is performed by first exposing the sample with aprehybridization solution. Next, the sample is incubated under bindingconditions for a suitable binding period with a sample solution that isto be analyzed. The sample solution generally contains a targetmolecule, e.g., a target nucleic acid, the presence or sequence of whichis of interest to the investigator. Binding conditions will varydepending on the application and are selected in accordance with thegeneral binding methods known including those referred to in: Maniatiset al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., ColdSpring Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology, Volume152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc.,San Diego, Calif.; Young and Davis (1983) Proc. Natl. Acad. Sci.(U.S.A.) 80: 1194, which are incorporated herein by reference. In someembodiments, the solution may contain about 1 molar of salt and about 1to 50 nanomolar of targets. Optionally, the fluid delivery systemincludes an agitator to improve mixing in the cavity, which shortens theincubation period. Finally, the sample is washed with a buffer, whichmay be 6× SSPE buffer, to remove the unbound targets. In someembodiments, the cavity is filled with the buffer after washing thesample.

Following hybridization and appropriate rinsing/washing, the cartridgedsubstrate may be aligned on a detection or imaging system, such as thosedisclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.) or U.S. patentapplication Ser. No. 08/195,889, filed Feb. 10, 1994, Ser. No.08/465,782, filed Jun. 6, 1995, Ser. No. 08/456,598, filed Jun. 1, 1995,incorporated herein by reference for all purposes. Such detectionsystems may take advantage of the cartridge's asymmetry (i.e., non-flushedge) by employing a holder to match the shape of the cartridgespecifically. Thus, the cartridge is assured of being properly orientedand aligned for scanning. The imaging systems are capable ofqualitatively analyzing the reaction between the probes and targets.Based on this analysis, sequence information of the targets isextracted.

VII. EXAMPLES Example-1 Comparison of Front-Side and Back-SidePhotolysis

Two substrate wafers were stripped, silanated and photoprotected. Thesubstrates were photolyzed through a mask having rectangular features of50 and 100 μm on the short side, for 13 half lives of thephotoprotecting group used. The first substrate was photolyzed from theback-side of the wafer, i.e., the synthesis surface was facing away fromthe photolyzing light source. The second substrate was photolyzed fromthe front-side, i.e., the synthesis surface was facing the light sourceand mask. Both substrates were then subjected to identical couplingreactions where a fluorescent 5′ protected phosphoramidite was coupledto the surface of the substrate.

FIGS. 9A and 9B illustrate the contrast difference between back-sideexposure synthesis and front-side exposure synthesis, respectively. FIG.9A shows a fluorescent scan of a substrate having fluorescent groupscoupled directly to the surface of the substrate using photolithographictechniques, with a mask having 50 μm and 100 μm feature sizes where theactivating light was shown through the back-side of the substrate. FIG.9B shows the same synthesis where the activation light was directed atthe front side of the substrate. The definition of the individualfeatures is greatly enhanced using this front-side photolysis.

FIGS. 9C and 9D provide a graphic illustration of the differences incontrast among features prepared using back-side vs. front-side methods.Specifically, the front-side exposure provides a much sharper contrastand greater feature definition. This greater definition permits a muchsmaller feature size by reducing bleed-over effects during exposure.While front-side exposure results in subjecting the synthesis surface toambient conditions during photolysis, this has not been found to haveany deleterious effects on the synthesis.

Example-2 Final Deprotection with Ethanolamine and Ethylenediamine

1-8 mer oligonucleotide probes were synthesized on glass substratesderivatized with bis (2-hydroxyethyl)aminopropyltriethoxysilane,according to standard protocols. In each case, ahexaethyleneglycol-based spacer phosphoramidite was coupled to thesurface before the oligonucleotide sequence, and a fluorescein-based“tag” phosphoramidite was coupled to the 5′ end of the oligonucleotides,usually in a checkerboard pattern. This allowed monitoring the loss ofprobes from the substrates, by ascertaining a decrease in the surfacefluorescence. The substrates were immersed in either concentratedaqueous ammonia or 50% ethanolic ethanolamine, or 50% ethanolicethylenediamine in sealed containers. At specific times, the substrateswere removed, washed with water, and the surface fluorescence was imagewas obtained, against a pH 7.2 phosphate buffer. After each scan, thesubstrates were washed again, dried in an inert atmosphere (N₂), andreturned to the deprotection solution. The surface fluorescence of thesubstrate immersed in the aqueous ammonia deprotection solution decayedwith a half-time of 8-10 hours. After two days in the ethanolic aminesolutions, only a 5% decay in surface fluorescence was observed.

Example-3 Comparison of Silanation Methods and Reagents

For comparison, glass substrates were derivatized with a number ofsilanes using solution-phase deposition methods. Mean functional surfacedensities were compared by fluorescent staining. Performance with regardto oligonucleotide synthesis was compared by synthesizing a 10mer probesequence on the substrates, deprotecting, and hybridizing them to astandard fluorescein labelled oligonucleotide target. Standardoligonucleotide synthesis cycles (couple-cap-oxidize) were used in allcases, but were modified slightly to allow for reagent delivery toflowcells for planar substrates.

The following silanes, obtained from Huls America were tested:

-   3-acetoxypropyltrimethoxysilane (“OAc”);-   3-glycidoxypropyltrimethoxysilane (“Epoxy”);-   4-(hydroxybutyramido)propyltriethoxysilane (“Mono”);-   3-aminopropyltriethoxysilane (“APS”); and-   3-N,N-bis (2-hydroxyethyl)aminopropyl triethoxysilane (“bis”)

Precleaned substrates were immersed in a 1% solution of the silane in 5%water, 95% ethanol, for 5 minutes with gentle agitation. The substrateswere then thoroughly rinsed with alcohol, dried under N₂, and cured at100° C. for 15 minutes. Prior to use, the acetoxypropyl-silanatedsubstrates were soaked in 50% ethanolic ethanolamine for 2 hours, thenrinsed and dried. Similarly, the glycidoxypropyl-silanated substrateswere soaked in 0.1 M aqueous HCl for 2 hours, rinsed then dried. Allother substrates were ised withoit further treatment.

The functional group density was then measured by fluorescent staining.Specifically, MeNPOC-hexaethyleneglycol-cyanoethl phosphoramidite wascoupled to the substrate and unreacvted sites were then capped with(MeO)₂PNiPr₂. A portion of the surface was illuminated through aphotolithographic mask for 300 seconds at 365 nm (15 mW/cm²) to removethe MeNPOC protecting groups. The free hydroxyls were then labeled witha fluorescein phosphoramidite (Fluoreprime™, Pharmacia Biotech). Thesubstrate was then deprotected n 50% ethanolic ethylenediamine andsurface fluorescence was measured with a scanning laser confocalmicroscope.

A 10mer oligonucleotide probe sequence (5′-TACCGTTCAG-3′) wassynthesized on a selected region of each substrate using light-directedsynthesis. After deprotection in 50% ethanolic ethylenediamine, thesubstrte was incubated in a solution of a complementaryfluorescein-labeled oligonucleotide target (10 nM oligonucleotide in5×SSPE buffer for 6 hours. After briefly washing the substrate once with5×SSPE, total surface-hybridized target oligonucleotide was quantitatedwith a scanning laser confocal microscope. Staining and hybridizationdata are summarized in FIG. 10 which illustrates effective silanation ofglass substrates using each of the above-described silane reagents.

Example-4 Direct Transfer of Protecting Groups to HydroxylatedSubstrates

Synthesis of MeNPOC-tetrazolide was carried out as follows: Tetrazole(7.0 g); 100 mmole) was combined with 17.5 ml of DIEA (13 g, 100 mmole)in 100 ml of THF, and a solution of 30 g (110 mmole) MeNPOC-chloride(See, Pease, et al, supra) in 100 ml THF was added dropwise over 20minutes while stirring under argon at 4° C. Stirring was continued foran additional hour at room temperature. 200 ml of hexane was then added.The precipitate was collected by filtration, redissolved in 200 ml DCMand washed 3 times with 0.05 M aqueous HCl to remove DIEA.HCL. Theorganic layer was dried with NaSO₄ and evaporated to obtain 24.5 g (80%)of the pure product, which was identified by ¹H-NMR, IR and massspectrometry.

MeNPOC-transfer to a hydroxylated substrate with MeNPOC-tetrazolide wascarried out as follows: Using methods described in the art, e.g., Peaseet al., supra, hydroxylated glass substrates were prepared by silanatingthe glass with bis-(hydroxyethyl)aminopropyltriethoxysilane, and thenadding a linker phosphoramidite(MeNPOC-hexaethyleneglycolcyanoethyl-phosphoramidite) to the substratesusing a standard couple-cap-oxidize cycle. The substrates were thenexposed to light (365 nm at 25 mW/cm² for 240 seconds) to remove theMeNPOC protecting groups from the linker. The free hydroxylated linkersubstrates were exposed to freshly mixed solutions of MeNPOC-tetrazolide(0.2M) in ACN containing 10% v/v 2,6 lutidine±5% w/v NMI or DMAPactivator. After varying periods of time, the MeNPOC-tetrazolidesolutions were removed and N,N-diisopropyl-dimethylphosphoramidite wasadded using the standard couple-cap-oxidize cycle in order to cap anyunreacted hydroxyl groups. To assess the extent of MeNPOC transfer, thesubstrate was photolysed again, and the reexposed hydroxyls were reactedwith a fluorescent phosphoramidite (Fluoreprime™, Pharmacia Biotech),added with the same couple-cap-oxidize protocol. The substrates werefinally deprotected with 50% ethanolic ethanolamine and the mean surfacefluorescence was measured with a laser scanning confocal microscope.FIG. 11 shows the extent of reprotection with MeNPOC tetrazolide as afunction of time and catalyst.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1. A method of forming an array of polymers on a surface of a substrate,comprising: providing a substrate having a first surface coated withfunctional groups protected with a photolabile protecting group, and asecond surface having a layer disposed thereon, said layer including oneor more of an index matching compound, a light absorbing compound and anantireflective compound; and sequentially activating and couplingmonomers in different selected regions of said substrate to form aplurality of different polymer sequences in different known locations onsaid surface of said substrate, wherein said activating step comprisesdirecting an activation radiation at said first surface of saidsubstrate. 2-49. (canceled)
 50. A method for synthesizing an array ofchemical compounds on the surface of a support wherein said synthesiscomprises a plurality of steps, said method comprising performing atleast two of said steps by: (a) placing a support having afunctionalized surface into a chamber of a flow cell and subjecting saidsurface to one step of said synthesis and (b) placing said support intoa chamber of another flow cell and subjecting said surface to anotherstep of said synthesis.
 51. A method according to claim 50 furthercomprising placing said support after step (b) of said synthesis into achamber of another flow cell and subjecting said surface to another stepof said synthesis.
 52. A method according to claim 50 wherein saidsynthesis comprises “n” number of steps and said method comprisesindependently placing a support into a chamber of one of “n” number offlow cells and subjecting said surface to a different step of saidsynthesis in each of said flow cells.
 53. A method according to claim 50wherein reagents for step (a) of said synthesis are in separate fluidcommunication with said flow cell of step (a) and reagents for step (b)of said synthesis are in separate fluid communication with said flowcell of step (b).
 54. A method according to claim 50 wherein at leastone of said steps of said synthesis comprises contacting said surfacewith a fluid reagent and washing said surface.
 55. A method according toclaim 50 wherein said chemical compounds are polymers.
 56. A methodaccording to claim 55 wherein said polymers are biopolymers.
 57. Amethod according to claim 50 wherein said flow cells comprise a holderfor said support.
 58. A method according to claim 50 wherein said flowcells comprise at least one inlet and an outlet.
 59. A method accordingto claim 58 wherein a condition of fluid exiting said outlet isdetermined and based on said determination at least a portion of saidfluid is directed to said inlet or to a waste container for said fluid.60. A method according to claim 58 wherein fluid exiting said outlet issubjected to purification and a condition of said fluid is determinedand based on said determination at least a portion of said fluid isdirected to said inlet or to a waste container for said fluid.
 61. Amethod according to claim 58 wherein a wash solution and a reagent forsaid synthesis are independently directed to said inlet.
 62. A methodfor synthesizing an array of biopolymers on the surface of a supportwherein said synthesis comprises a plurality of monomer additions, saidmethod comprising after each of said monomer additions (a) placing saidsupport into a chamber of a flow cell and subjecting said surface to astep of said synthesis that is subsequent to a monomer addition and (b)placing said support into a chamber of another flow cell and subjectingsaid surface to another step of said synthesis that is subsequent tostep (a).
 63. A method according to claim 62 wherein each of said stepscomprises a wash.
 64. A method according to claim 62 wherein saidbiopolymers are polynucleotides.
 65. A method according to claim 62wherein step (a) comprises subjecting said surface to an oxidizingagent.
 66. A method according to claim 62 wherein step (b) comprisessubjecting said surface to an agent for removing a protecting group. 67.A method according to claim 62 wherein said flow cells comprise at leastone inlet and an outlet and a holder for said support.
 68. A methodaccording to claim 62 wherein a condition of fluid exiting said flowcell is determined and based on said determination at least a portion ofsaid fluid is directed to said flow cell or to a waste container forsaid fluid.
 69. A method according to claim 67 wherein fluid exitingsaid flow cell is subjected to purification and a condition of saidfluid is determined and based on said determination at least a portionof said fluid is directed to said flow cell or to a waste container forsaid fluid.
 70. A method according to claim 67 wherein a wash solutionand a reagent for said synthesis are independently directed to saidinlet.
 71. A method according to claim 67 wherein said fluid is anorganic solvent.
 72. A method according to claim 62 wherein saidbiopolymers are peptides.
 73. 24. A method according to claim 62 whereinsaid biopolymers are synthesized on said surface in multiple arrays andsaid support is subsequently diced into individual arrays of biopolymerson a support.
 74. A method according to claim 62 wherein reagents forsaid first step of said synthesis are in separate fluid communicationwith said first flow cell and reagents for said second step of saidsynthesis are in separate fluid communication with said second flowcell.
 75. A method according to claim 62 further comprising exposing thearray to a sample and reading the array.
 76. A method according to claim75 comprising forwarding data representing a result obtained from areading of the array.
 77. A method according to claim 76 wherein thedata is transmitted to a remote location.
 78. A method according toclaim 75 comprising receiving data representing a result of aninterrogation obtained by the reading of the array.
 79. An apparatus forsynthesizing an array of biopolymers on the surface of a support, saidapparatus comprising: (a) a plurality of flow cells, (b) one or morefluid dispensing stations in fluid communication with one or more ofsaid plurality of flow cells, (c) a station for monomer addition to saidsurface of said support, and (d) a mechanism for moving a support to andfrom said station for monomer addition and a flow cell and from one flowcell to another flow cell.
 80. An apparatus according to claim 79further comprising a controller for controlling the movement of saidmechanism.
 81. An apparatus according to claim 79 wherein said mechanismis a robotic arm.
 82. An apparatus according to claim 79 wherein saidflow cells comprise a chamber, a holder for said support, at least oneinlet and an outlet.
 83. An apparatus according to claim 82 wherein saidapparatus further comprises a manifold in fluid communication with saidinlet.
 84. An apparatus according to claim 79 wherein said apparatusfurther comprises a purification system in fluid communication with saidoutlet.
 85. An apparatus according to claim 79 wherein said apparatusfurther comprises a holding chamber in fluid communication with saidpurification system.
 86. An apparatus according to claim 85 wherein saidapparatus further comprises a sensor in fluid communication with holdingchamber.
 87. An apparatus according to claim 79 wherein said apparatusfurther comprises a sensor in fluid communication with said outlet. 88.An apparatus according to claim 79 wherein reagents for a step of saidsynthesis are in separate fluid dispensing stations in fluidcommunication with one of said flow cells and reagents for another stepof said synthesis are in separate fluid dispensing stations in fluidcommunication with another of said flow cells.
 89. A method comprisingusing an array, prepared by an apparatus according to claim 79, byexposing the array to a sample and reading the array.
 90. A methodaccording to claim 89 comprising forwarding data representing a resultobtained from a reading of the array.
 91. A method according to claim 90wherein the data is transmitted to a remote location.
 92. A methodaccording to claim 89 comprising receiving data representing a result ofan interrogation obtained by the reading of the array.
 93. An apparatusfor synthesizing an array of biopolymers on the surface of a support,said apparatus comprising: (a) a plurality of flow cells wherein saidflow cells comprise a chamber, a holder for said support, at least oneinlet and an outlet, wherein each of said inlets is in fluidcommunication with a manifold and wherein said outlet is in controlledfluid communication with one or more purification systems, holdingchambers and sensors, (b) one or more fluid dispensing stations in fluidcommunication with one or more of said plurality of flow cells by meansof said manifolds, (c) a station for monomer addition to said surface ofsaid support, (d) a mechanism for moving a support to and from saidstation for monomer addition and a flow cell and from one flow cell toanother flow cell, and (e) a controller for controlling the movement ofsaid mechanism.
 94. An apparatus according to claim 93 wherein saidmechanism is a robotic arm.
 95. An apparatus according to claim 93wherein said apparatus further comprises a purification system in fluidcommunication with said outlet.
 96. An apparatus according to claim 93wherein said apparatus further comprises a holding chamber in fluidcommunication with said purification system.
 97. An apparatus accordingto claim 96 wherein said apparatus further comprises a sensor in fluidcommunication with holding chamber.
 98. An apparatus according to claim93 wherein said apparatus further comprises a sensor in fluidcommunication with said outlet.